Brain-associated inhibitor of tissue-type plasminogen activator

ABSTRACT

The present invention relates to a novel BAIT protein which is a member of serpin superfamily which is expressed primarily in brain tissue. In particular, isolated nucleic acid molecules are provided encoding the human and recombinant methods for producing the same. The invention further relates to screening methods for identifying agonists and antagonists of BAIT activity. Also provided are diagnostic methods for detecting nervous system-related disorders and therapeutic methods for treating nervous system-related disorders. Additionally, the present invention is related to methods of treating patients with BAIT polynucleotides or polypeptides, wherein said patients have had a stroke.

[0001] This application claims benefit of 35 U.S.C. section 119(e) basedon copending U.S. Provisional Application Serial No. 60/123,704, filedMar. 10, 1999, herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to a novel human gene encoding apolypeptide expressed in human brain tissue which is a member of theserine protease inhibitor (“serpin”) superfamily and appears to be ahuman homolog of “neuroserpin,” a serpin recently identified in thechicken. More specifically, isolated nucleic acid molecules are providedencoding a human polypeptide named Brain-Associated Inhibitor ofTissue-Type Plasminogen Activator, hereinafter referred to as “BAIT.”BAIT polypeptides are also provided, as are vectors, host cells andrecombinant methods for producing the same. The invention furtherrelates to screening methods for identifying agonists and antagonists ofBAIT activity. Also provided are diagnostic methods for detectingdisorders related to the central and peripheral nervous system and thecirculatory system, and therapeutic methods for treating such disorders.

BACKGROUND OF THE INVENTION

[0003] Localized proteolytic activity through the action of proteasesplays a critical regulatory role in a variety of important biologicalprocesses. For instance, the enzyme plasmin plays such a role inhemostasis, angiogenesis, tumor metastisis, cellular migration andovulation. Plasmin is generated from its precursor zymogen plasminogenby the action of plasminogen activators (PAs) such as tissue-type PA(t-PA) and urokinase-type (u-PA), both of which are serine proteases.The activity of the PA system is precisely regulated by severalmechanisms, one of which involves the interaction of t-PA and u-PA withspecific plasminogen activator inhibitors. Among these serine proteaseinhibitors (i.e., serpins), plasminogen activator inhibitor type I(PAI-1) is unique in its ability to efficiently inhibit u-PA as well asthe single and two-chain forms of t-PA. High PAI-1 levels are associatedwith an increased risk of thromboembolic disease, while PAI-1 deficiencymay represent an inherited autosomal recessive bleeding disorder. See,for instance, Reilly, T. M., et al., Recombinant plasminogen activatorinhibitor type 1: a review of structural, functional, and biologicalaspects, Blood Coag. And Fibrinolysis 5:73-81 (1994).

Serpin Mechanism

[0004] The serpins are a gene family that encompasses a wide variety ofprotein products, including many of the proteinase inhibitors in plasma(Huber, R. (1989) Biochemistry, 28, 8951-8966). However, in spite oftheir name, not all serpins are proteinase inhibitors. They includesteroid binding globulins, the prohormone angiotensinogen, the egg whiteprotein ovalbumin, and barley protein Z, a major constituent of beer.The serpins are thought to share a common tertiary structure (Doolittle,R. F. (1983) Science, 222, 417-419) and to have evolved from a commonancestor (Hunt, L. T (1980) Biochemical and Biophysical ResearchCommunications, 95, 864-871). Proteins with recognizable sequencehomology have been identified in vertebrates, plants, insects andviruses but not, thus far, in prokaryotes (Huber, R. (1989)Biochemistry, 28, 8951-8966; Sasaki, T. (1991) Eur J Biochem, 202,255-261; Komiyama, T., (1994) The Journal of Biological Chemistry, 269,19331-19337). Current models of serpin structure are based largely onseminal X-ray crystallographic studies of one member of the family,(α1-antitrypsin (α1AT), also called (α1-proteinase inhibitor (Huber, R.(1989) Biochemistry, 28, 8951-8966). The structure of a modified form ofα1AT, cleaved in its reactive center, was solved by Loebermann andcoworkers in 1984 (Loebermann, H., et. al. (1984) J Mol Biol, 177,531-557). An interesting feature of this structure was that the tworesidues normally comprising the reactive center (Met-Ser), were foundon opposite ends of the molecule, separated by almost 70 Å. Loebermannand coworkers proposed that a relaxation of a strained configurationtakes place upon cleavage of the reactive center peptide bond, ratherthan a major rearrangement of the inhibitor structure. In this model,the native reactive center is part of an exposed loop, also called thestrained loop (Loebermann, H., et. al. (1984) J Mol Biol, 177, 531-557;Carrell, R. W., & Boswell, D. R. (1986) In A. J. Barrett & G. Salvesen(Eds.), Proteinase Inhibitors. (pp. 403-420). Amsterdam: ElsevierScience Publishers (Biomedical Division); Sprang, S. R. (1992) TrendsBiochem Sci, 17, 49-50). Upon cleavage, this loop moves or “snaps back”,becoming one of the central strands in a major β-sheet structure(β-sheet A). This transformation is accompanied by a large increase inthermal stability (Carrell, R. W., & Owen, M. C. (1985) Nature, 317,730-732; Gettins, P., & Harten, B. (1988) Biochemistry, 27, 3634-3639;Bruch, M., Weiss, V., & Engel, J. (1988) The Journal of BiologicalChemistry, 263, 16626-16630; Lawrence, D. A., et. al. (1994) The Journalof Biological Chemistry, 269, 27657-27662).

[0005] Recent crystallographic structures of several native serpins,with intact reactive center loops, have confirmed Loebermann'shypothesis that the overall native serpin structure is very similar tocleaved α1AT, but that the reactive center loop is exposed above theplane of the molecule (Schreuder, H. A., et. al. (1994) NatureStructural Biology, 1, 48-54; Carrell, R. W. et al. (1994) Structure, 2,257-270; Stein, P. E., et. al. (1990) Nature, 347, 99-102; Wei, A., et.al. (1994) Nature Structural Biology, 1, 251-258). Additional evidencefor this model has come from studies where synthetic peptides,homologous to the reactive center loops of α1AT, antithrombin III(ATIII), or PAI-1 when added in trans, incorporate into their respectivemolecules, presumably as a central strand of β-sheet A (Björk, I., et.al. (1992) The Journal of Biological Chemistry, 267, 1976-1982; Björk,I., et. al. (1992) The Journal of Biological Chemistry, 267,19047-19050; Schulze, A. J., et. al. (1990) Eur J Biochem, 194, 51-56;Carrell, R. W., Evans, D. L., & Stein, P. E. (1991) Nature, 353,576-578; Kvassman, (1995) J Biol Chem, 270, 27942-27947). This leads toan increase in thermal stability similar to that observed followingcleavage of a serpin at its reactive center, and converts the serpinfrom an inhibitor to a substrate for its target proteinase. A thirdserpin structural form has also been identified, the so-called latentconformation. In this structure the reactive center loop is intact, butinstead of being exposed, the entire amino-terminal side of the reactivecenter loop is inserted as the central strand into β-sheet A (Mottonen,J., et. al. (1992) Nature, 355, 270-273). This accounts for theincreased stability of latent PAI-1 (Lawrence, et. al. (1994)Biochemistry, 33, 3643-3648) as well as its lack of inhibitory activity(Hekman, C. M., & Loskutoff, D. J. (1985) The Journal of BiologicalChemistry, 260, 11581-11587). The ability to adopt this conformation isnot unique to PAI-1, but has also now been shown for ATIII and β1AT(Carrell, R. W. et al. (1994) Structure, 2, 257-270; Lomas, D. A., et.al. (1995) J Biol Chem, 270, 5282-5288). Together, these data have ledto the hypothesis that active serpins have mobile reactive center loops,and that this mobility is essential for inhibitor function (Lawrence, D.A., et. al. (1990) The Journal of Biological Chemistry, 265,20293-20301; Carrell, R. W., Evans, D. L., & Stein, P. E. (1991) Nature,353, 576-578; Carrell, R. W., & Evans, D. L. I. (1992) Curr Opin StructBiol, 2, 438-446; Lawrence, D. A., et. al. (1994) The Journal ofBiological Chemistry, 269, 27657-27662; Shore, J. D., et. al. (1994) TheJournal of Biological Chemistry, 270, 5395-5398; Lawrence, D. A., et.al. (1995) J. Biol Chem, 270, 25309-25312; Fa, M., et. al., (1995)Biochemistry, 34, 13833-13840; Olson, S. T., et. al. (1995) J Biol Chem,270, 30007-30017). The large increase in thermal stability observed withloop insertion, is presumably due to reorganization of the five strandedβ-sheet A from a mixed parallel-antiparallel arrangement to a sixstranded, predominantly antiparallel β-sheet (Carrell, R. W., & Owen, M.C. (1985) Nature, 317, 730-732; Gettins, P., & Harten, B. (1988)Biochemistry, 27, 3634-3639; Bruch, M., Weiss, V., & Engel, J. (1988)The Journal of Biological Chemistry, 263, 16626-16630; Lawrence, et. al.(1994) Biochemistry, 33, 3643-3648). This dramatic stabilization has ledto the suggestion that native inhibitory serpins may be metastablestructures, kinetically trapped in a state of higher free energy thantheir most stable thermodynamic state (Lawrence, D. A., et. al. (1995)J. Biol Chem, 270, 25309-25312; Lee, K. N., et. al. (1996) NatureStructural Biology, 3, 497-500). Such an energetically unfavorablestructure would almost certainly be subject to negative selection, andthus its retention in all inhibitory serpins implies that it has beenconserved for functional reasons.

[0006] The serpins act as “suicide inhibitors” that react only once witha target proteinase forming an SDS-stable complex. They interact bypresenting a “bait” amino acid residue, in their reactive center, to theenzyme. This bait residue is thought to mimic the normal substrate ofthe enzyme and to associate with the specificity crevice, or S1 site, ofthe enzyme (Carrell, R. W., & Boswell, D. R. (1986) In A. J. Barrett &G. Salvesen (Eds.), Proteinase Inhibitors. (pp. 403-420). Amsterdam:Elsevier Science Publishers (Biomedical Division); Huber, R. (1989)Biochemistry, 28, 8951-8966; Bode, W., & Huber, R. (1994) Fibrinolysis,8, 161-171.). The bait amino acid is called the P1 residue, with theamino acids toward the N-terminal side of the scissile reactive centerbond labeled in order P1 P2 P3 etc. and the amino acids on the carboxylside labeled P1′ P2′ etc. (Carrell, R. W., & Boswell, D. R. (1986) In A.J. Barrett & G. Salvesen (Eds.), Proteinase Inhibitors. (pp. 403-420).Amsterdam: Elsevier Science Publishers (Biomedical Division)). Thereactive center P1-P1′ residues, appear to play a major role indetermining target specificity. This point was dramatically illustratedby the identification of a unique human mutation, α1AT “Pittsburgh”, inwhich a single amino acid substitution of Arg for Met at the P1 residueconverted α1AT from an inhibitor of elastase to an efficient inhibitorof thrombin, resulting in a unique and ultimately fatal bleedingdisorder (Owen, M. C., et. al. (1983) N Engl J Med, 309, 694-698).Numerous mutant serpins have been constructed, demonstrating a widerange of changes in target specificity, particularly with substitutionsat P1 (York, J. D., et. al. (1991) The Journal of Biological Chemistry,266, 8495-8500; Strandberg, L., et. al. (1991) The Journal of BiologicalChemistry, 266, 13852-13858; Shubeita, H. E., et. al. (1990) The Journalof Biological Chemistry, 265, 18379-18385; Lawrence, D. A., et. al.(1990) The Journal of Biological Chemistry, 265, 20293-20301; Sherman,P. M., et. al., (1992) The Journal of Biological Chemistry, 267,7588-7595).

[0007] The exact structure of the complex between serpins and theirtarget proteinases has been controversial. Originally it was thoughtthat the complex was covalently linked via an ester bond between theactive site serine residue of the proteinase and the newcarboxyl-terminal end of the P1 residue, forming an acyl-enzyme complex(Moroi, M., & Yamasaki, M. (1974) Biochim Biophys Acta, 359, 130-141;Owen, W. G. (1975) Biochim Biophys Acta, 405, 380-387; Cohen, A. B., etal., (1977) Proceedings of the National Academy of Sciences, USA, 74,4311-4314; Nilsson, T., & Wiman, B. (1982) FEBS Lett, 142, 111-114).However, in the late 1980s and early 1990s it was suggested that thisinterpretation was incorrect, and that the serpin-proteinase complex isinstead trapped in a tight non-covalent association similar to the socalled standard mechanism inhibitors of the Kazal and Kunitz family(Longstaff, C., & Gaffney, P., J. (1991) Biochemistry, 30, 979-986;Shieh, B. H., et. al. (1989) J Biol Chem, 264, 13420-13423; Potempa, J.,et. al. (1994) The Journal of Biological Chemistry, 269, 15957-15960).Alternatively, one study suggested a hybrid of these two models wherethe complex was frozen in a covalent but un-cleaved tetrahedraltransition state configuration (Matheson, N. R., et. al. (1991) TheJournal of Biological Chemistry, 266, 13489-13491). Recently however,new data by several groups have suggested that the debate has come fullcircle, with various studies using independent methods indicating thatthe inhibitor is indeed cleaved in its reactive-center and that thecomplex is most likely trapped as a covalent acyl-enzyme complex(Lawrence, D. A., et. al. (1995) J. Biol Chem, 270, 25309-25312; Olson,S. T., et. al. (1995) J Biol Chem, 270, 30007-30017; Fa, M., et. al.,(1995) Biochemistry, 34, 13833-13840; Wilczynska, M., et. al. (1995) TheJournal of Biological Chemistry, 270, 29652-29655; Lawrence, D. A., et.al. (1994) The Journal of Biological Chemistry, 269, 27657-27662; Shore,J. D., et. al. (1994) The Journal of Biological Chemistry, 270,5395-5398; Plotnick, M. I., et. al. (1996) Biochemistry, 35, 7586-7590).

[0008] Recently, three groups have almost simultaneously proposedsimilar mechanisms for serpin inhibition (Lawrence, D. A., et. al.(1995) J. Biol Chem, 270, 25309-25312; Wilczynska, M., et. al. (1995)The Journal of Biological Chemistry, 270, 29652-29655; Wright, H. T., &Scarsdale, J. N. (1995) Proteins, 22,210-225). This model suggests thatupon encountering a target proteinase, a serpin binds to the enzymeforming a reversible complex that is similar to a Michaelis complexbetween an enzyme and substrate. Next, the proteinase cleaves the P1-P1′peptide bond resulting in formation of a covalent acyl-enzymeintermediate. This cleavage is coupled to a rapid insertion of thereactive center loop (RCL) into β-sheet A at least up to the P9position. Since the RCL is covalently linked to the enzyme via theactive-site Ser, this transition should also affect the proteinase,significantly changing its position relative to the inhibitor. If,during this transition, the RCL is prevented from attaining fullinsertion because of its association with the enzyme, and the complexbecomes locked, with the RCL only partially inserted, then the resultingstress might be sufficient to distort the active site of the enzyme.This distortion would then prevent efficient deacylation of theacyl-enzyme intermediate, thus trapping the complex. However, if RCLinsertion is prevented, or if deacylation occurs before RCL insertionthen the cleaved serpin is turned over as a substrate and the activeenzyme released. This means that what determines whether a serpin is aninhibitor or a substrate is the ratio of k_(diss) to k_(Stab) Ifdeacylation (k_(diss)) is faster than RCL insertion (k_(stab)) then thesubstrate reaction predominates. However, if RCL insertion anddistortion of the active site can occur before deacylation then thecomplex is frozen as a covalent acyl-enzyme. A similar model was firstproposed-in 1990 (Lawrence, D. A., et. al. (1990) The Journal ofBiological Chemistry, 265, 20293-20301) and is consistent with studiesdemonstrating that RCL insertion is not required for proteinase bindingbut is necessary for stable inhibition (Lawrence, D. A., et. al. (1994)The Journal of Biological Chemistry, 269, 27657-27662) as well as theobservation that only an active enzyme can induce RCL insertion (Olson,S. T., et. al. (1995) J Biol Chem, 270, 30007-30017). Very recently,direct evidence for this model was provided by Plotnick et al., who byNMR observed an apparent distortion of an enzyme's catalytic site in aserpin-enzyme complex (Plotnick, M. I., et. al. (1996) Biochemistry, 35,7586-7590). In conclusion, these data suggest that serpins act asmolecular springs where the native structure is kinetically trapped in ahigh energy state. Upon association with an enzyme some of the energyliberated by RCL insertion is used to distort the active site of theenzyme, preventing deacylation and trapping the complex.

Nervous System

[0009] During the development of the nervous system, neurons form axonswhich extend along a prespecified path into the target area, where theyengage in the formation and refinement of synaptic connections. Thesestages depend critically on the capability of the axonal growth cones tointeract with a variety of structures which they encounter along theirway and at their destination. These structures include cell surfaces ofneuronal and non-neuronal origin and the extracellular matrix. Alongtheir trajectory and at their target sites, growth cones not onlyreceive and respond to signals from their local environment, but alsoactively secrete macromolecules. In particular, secreted proteases havebeen implicated in supporting the growth cone advancement through thetissue. More than a decade ago, it was demonstrated that plasminogenactivators are axonally secreted by neurons in culture. Recently, theiroccurrence in the developing rat nervous system during the period ofaxon outgrowth has been revealed. Moreover, several pieces of evidencewere presented which indicated that serine proteases, such asplasminogen activators or thrombin, are involved in restructuring of thesynaptic connectivity during development and regeneration. Suchprocesses include elimination during development and synaptic plasticityassociated with learning and memory in the adult. See, for instance,Osterwalder, T., et al., “Neuroserpin, an axonally secreted serineprotease inhibitor,” EMBO J. 15:2944-2953 (1996).

[0010] During normal development of the nervous system, about 50% ofpostmitotic lumbosacral motoneurons undergo naturally occurring(programmed) cell death during a period when these cells are formingsynaptic connections with their target muscles. Naturally occurringmotoneuron death has been described in many vertebrate species,including chicken, mouse, rat, and human embryos or fetuses. Forexample, programmed motoneuron death occurs between embryonic day (E)6and E10 in the chicken. This system has been used as a biological modelfor testing different neurotrophic agents on motoneuron survival invivo. See, for instance, Houenou, L. J., et al., “A serine proteaseinhibitor, protease nexin I, rescues motoneurons from naturallyoccurring and axotomy-induced cell death,” Proc. Natl. Acad. Sci. USA92:895-899 (1995).

[0011] Although programmed cell death is completed before birth inmammals, the maintenance of motoneurons continues to be dependent onsupport from the target for some time after birth. Thus, if transectionof motor axons is performed in neonatal mammals and reinnervation isprevented, a large number of motoneurons degenerate and die.Axotomy-induced death of motoneurons has also been extensively used as amodel for testing the survival effects of various agents, includingneurotrophic and growth factors on motoneurons.

[0012] Protease nexin I (PNI), also known as glia-derived nexin, is a43-47-kDa protein that was first found secreted by cultured fibroblastsbut is also produced by glial (glioma and primary) and skeletal musclecells. PNI has been shown to promote neurite outgrowth from differentneuronal cell types. These include neuroblastoma cells, as well asprimary hippocampal and sympathetic neurons. The neurite promotingactivity of PNI in vitro is mediated by inhibition of thrombin, a potentserine protease. PNI (mRNA and protein) is transiently up-regulated inrat sciatic nerve after axotomy, and PNI-producing cells are localizeddistal to the lesion site. This up-regulation of PNI occurs 2-3 daysafter a similar up-regulation of prothrombin and thrombin in the distalstump. Free PNI protein is significantly decreased, while endogenousPNI-thrombin complexes are increased, in various anatomical brainregions, including hippocampus of patients with Alzheimer disease. Whenconsidered together with the recent demonstration that PNI can promotethe in vitro survival of mixed mouse spinal chord neurons and that PNIis released from glia cells by neuropeptides such as vasoactiveintestinal polypeptide, these observations suggest that PNI may play aphysiological role in neuronal survival, differentiation, and/or axonalregeneration in vivo.

[0013] Recently, it has been reported that PNI rescues spinal motoneurondeath in the neonatal mouse. Houenou, L. J. et al., 1995, supra. Thesurvival effect of PNI on motoneurons during the period of programmedcell death was not associated with increased intramuscular nervebranching. PNI also significantly increased the nuclear size ofmotoneurons during the period of programmed cell death and preventedaxotomy-induced atrophy of surviving motoneurons. These results indicatea possible role of PNI as a neurotrophic agent. They also support theidea that serine proteases or, more precisely, the balance of proteasesand serpins may be involved in regulating the fate of neuronal cellsduring development.

[0014] More recently, a cDNA encoding an axonally secreted glycoproteinof central nervous system (CNS) and peripheral nervous system (PNS)neurons of the chicken has been cloned and sequenced. Osterwalder, T.,et al., 1996) supra. Analysis of the primary structural featurescharacterized this protein as a novel member of the serpin superfamilywhich was therefore called “neuroserpin.” No demonstration of inhibitionof any protease was included in this report, however. In situhybridization revealed a predominately neuronal expression during thelate stages of neurogenesis and in the adult brain in regions whichexhibit synaptic plasticity. Thus, it has been suggested thatneuroserpin may function as an axonally secreted regulator of the localextracellular proteolysis involved in the reorganization of the synapticconnectivity during development and synapse plasticity in the adult. Arole for serine proteases and serpins in neuronal remodeling is furthersupported by the finding that elevated tPA mRNA and protein levels arefound in cerebellar Purkinje neurons of rats undergoing motor learning(Seeds N W; Williams B L; Bickford P. C., “Tissue plasminogen activatorinduction in Purkinje neurons after cerebellar motor learning.” Science270:1992-4 (1995)).

[0015] The amplification of a human cDNA fragment of about 450 bpcorresponding to the region of the chicken cDNA encoding the putativereactive site loop of the so- called neuroserpin, using a polymerasechain reaction with two pairs of nested primers flanking that region,has also been reported. Osterwalder, T., et al., 1996, supra, page 2946.The authors also reported that the deduced amino acid sequences of thehuman and corresponding mouse cDNA exhibited a sequence identity of 88%and 87% respectively, with chicken neuroserpin. However, the human DNAsequence in a related serpin derived from human hypothalamus isdescribed in WO96/40922 published Dec. 19, 1996 is about 99% the same asthe present invention.

[0016] Thus, there is a need for human polypeptides that function asserpins in the regulation of various serine proteases, particularly inthe nervous system, since disturbances of such regulation may beinvolved in disorders relating to hemostasis, angiogenesis, tumormetastisis, cellular migration and ovulation, as well as neurogenesis;and, therefore, there is a need for identification and characterizationof such human polypeptides which can play a role in preventing,ameliorating or correcting such disorders.

[0017] A related serpin (CAPE) derived from human hypothalamus isdescribed in WO96/40922 published Dec. 19, 1996. This published CAPEserpin differs from the BAIT of the present invention by having 17 ofits CAPE amino acids replaced by 23 different BAIT amino acids.Specifically when numbering from the first methionine, HGS Alamine (27)is replaced by CAPE Valine; HGS Aspartic Acid (173) replaces an unknownCAPE amino acid; the six HGS aminoacids 319-324 are replaced in CAPE by5 different amino acids, and the 15 HGS amino acids 351-365 are replacedby only 10 CAPE amino acids. Thus the BAIT of the present inventioncontains 23 amino acids in 4 locations that are not found in the CAPEpolypeptide.

Stroke

[0018] Stroke is the second most common cause of death in the worldafter heart disease and a leading cause of disability. The World HealthReport 1999: Making A Difference. 1-121. 1999. Geneva, Switzerland, TheWorld Health Organization. It is estimated that in United States thereis a stroke approximately every minute and a person dies of stroke aboutevery 3.5 minutes. Thorvaldsen P, et. al. Stroke (1995) 26:361-367.Various strategies have been employed to reduce stroke morbidity andmortality, one of which has been thrombolysis with tPA in order torestore cerebral blood flow to ischemic brain tissue. Thrombolysis withtPA produces arterial recanalization in 40-67% of patients (Caplan L R,et. al. N.Engl.J Med. 1997;337: 1309-1310), and is associated withabsolute improvement in neurological function after 90 days in 12% ofthe patients treated within 3 hours of the onset of symptoms. N.Engl.JMed. 1995;333:1581-1587. However, in seeming contradiction to theseresults, animal studies have demonstrated that tPA-deficient mice have a41% decrease in stroke size and a 61% increase in neuronal survivalcompared with wild type animals following middle cerebral arteryocclusion. Wang Y F, et. al. Nat Med. 1998;4:228-231. Although theseresults have been recently challenged (Tabrizi P., et al.Arterioscler.Thromb.Vasc.Biol. 1999; 19:2801-2806; Klein G M, et al.Neurology 1999;52:1381-1384) they have also been reproduced by others.Nagai N, et al. Circulation 1999;99:2440-2444. Furthermore, this latterstudy also demonstrated that animals deficient in plasminogen had anincrease in stroke volume, while animals deficient in the primaryplasmin inhibitor, alpha2-antiplasmin, had a decrease in stroke sizesimilar to tPA null mice, suggesting a plasminogen-independent functionfor tPA in cerebral ischemia.

[0019] In both rats and mice, tPA expression is increased by events thatrequire neuronal plasticity, such as synaptic remodeling, long termpotentiation, kindling and seizures. Qian Z, et. al. Nature1993;361:453-457; Seeds N W, et. al. Science 1995;270:1992-1994; CarrollP M, et. al. Development 1994;120:3173-3183. Expression of tPA is alsocorrelated with CNS development and maintenance, and in the modulationof cell-cell and cell-extracellular matrix interactions. As in stroke,tPA-deficient mice are protected from excitotoxin-induced neuronaldeath. Friedman G C, Brain Res.Dev.Brain Res. 1994; 81:41-49; Ware J H,et. al. Brain Res.Bull. 1995;37:275-281. However, in contrast to stroke,plasminogen-deficient mice are also protected from excitotoxic injury(Ware J H, et. al. Brain Res.Bull. 1995;37:275-281; Sappino A P, et. al.J.Clin.Invest. 1993;92:679-685; Tsirka S E, et. al. Nature1995;377:340-344; Tsirka S E, et. al. Nature 1996;384:123-124; Tsirka SE, et. al. J.Neurosci. 1997;17:543-552) and it has been suggested thattPA and plasminogen may promote excitotoxin-induced neuronal deaththrough proteolysis of the neuronal extracellular matrix (ECM). Chen Z LCell 1997;91:917-925.

[0020] Following stroke there is a densely ischemic area where neuronsare irreversibly damaged, surrounded by an area known as “ischemicpenumbra”, where cerebral blood flow is sufficiently decreased toabolish electrical potentials yet sufficient to allow maintenance ofmembrane potentials and cellular ionic homeostasis. Symon L., ActaNeurol.Scand.Suppl. 1980;78:175-90:175-190; Hakim A M, Can.J Neurol.Sci.1987;14:557-559; Hossmann K A, Ann.Neurol. 1994;36:557-565. This zone ofpenumbra has also been observed in magnetic resonance image studies(MRI) of rats in the area surrounding the necrotic core. Pierce A R, et.al. J Cereb.Blood Flow Metab. 1997;17:183-190; Grohn O H, et al., JCereb.Blood Flow Metab. 1998;18:911-920; Hoehn-Berlage M, et. al., JCereb.Blood Flow Metab. 1995;15:1002-1011. With time, this potentiallysalvageable area of penumbra, or reversible ischemia, tends to becomeinfarcted. In vivo microdialysis has demonstrated that after cerebralischemia there is a large release of excitotoxins (Benveniste H, et.al., J Neurochem. 1984;43:1369-1374; Globus M Y, J Neurochem.1988;51:1455-1464; Miyashita K, et. al., Neuroreport. 1994;5:945-948;Uchiyama-Tsuyuki Y, et. al., J Neurochem. 1994;62:1074-1078) not only inthe infarcted core but also in the area of ischemic penumbra (Takagi K,et. al. i Cereb.Blood Flow Metab. 1993;13:575-585), where the presenceof apoptotic cells has also been described. Li Y, et. al. J Cereb.BloodFlow Metab. 1995;15:389-397; Linnik M D, et. al., Stroke1993;24:2002-2008; Linnik M D, et. al., Brain Res.Mol.Brain Res.1995;32:116-124. Since tPA may play an significant role in bothexcitotoxin- and ischemia-induced neuronal degeneration, then it ispossible that an inhibitor of tPA might play an important role inneuronal survival after stroke.

SUMMARY OF THE INVENTION

[0021] The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding the human BAIT polypeptide havingthe amino acid sequence shown in FIG. 1 (SEQ ID NO:2) or the amino acidsequence encoded by the cDNA clone deposited in a bacterial host as ATCCDeposit Number 97722 on September 18, 1996. The nucleotide sequencedetermined by sequencing the deposited BAIT clone, which is shown inFIG. 1 (SEQ ID NO:1), contains an open reading frame encoding a completepolypeptide of 410 amino acid residues, including an initiation codon atpositions 89-91, and a predicted molecular weight of about 46.4 kDa. Theencoded polypeptide has a leader sequence of 18 amino acids, underlinedin FIG. 1; and the amino acid sequence of the expressed mature BAITprotein is also shown in FIG. 1, as amino acid residues 19-410 (SEQ IDNO:2).

[0022] The human BAIT protein of the present invention has been shown toexhibit selective inhibition of tissue-type plasminogen activator (t-PA)with relatively little inhibition of trypsin, thrombin or urokinase-typeplasminogen activator (u-PA). The human BAIT polypeptide also sharesextensive sequence homology with the translation product of the mRNA fora serpin-related protein isolated from brain cDNA library which has beennamed “neuroserpin” (SEQ ID NO:3) (see FIG. 2). As noted above,neuroserpin in the chicken is thought to play an important role inregulation of local extracellular proteolysis involved in thereorganization of the synaptic connectivity during development andsynapse plasticity in the adult. The homology between neuroserpinand-BAIT (90% amino acid similarity) indicates that BAIT also may play asimilar role in neurogenesis in humans.

[0023] Thus, one aspect of the invention provides an isolated nucleicacid molecule comprising a polynucleotide having a nucleotide sequenceselected from the group consisting of: (a) a nucleotide sequenceencoding the BAIT polypeptide having the complete amino acid sequence inFIG. 1 (SEQ ID NO:2); (b) a nucleotide sequence encoding the expressedmature BAIT polypeptide having the amino acid sequence at positions19-410 in FIG. 1 (SEQ ID NO:2); (c) a nucleotide sequence encoding theBAIT polypeptide having the complete amino acid sequence encoded by thecDNA clone contained in ATCC Deposit No. 97722; (d) a nucleotidesequence encoding the mature BAIT polypeptide having the amino acidsequence encoded by the cDNA clone contained in ATCC Deposit No. 97722;and (e) a nucleotide sequence complementary to any of the nucleotidesequences in (a), (b), (c) or (d) above.

[0024] Further embodiments of the invention include isolated nucleicacid molecules that comprise a polynucleotide having a nucleotidesequence at least 99.1% identical, and more preferably at least 99.2%,99.3%, 99.4%, 99.5%, 99.6.%, 99.7%, 99.8% or 99.9% identical, to any ofthe nucleotide sequences in (a), (b), (c), (d) or (e), above, or apolynucleotide which hybridizes under stringent hybridization conditionsto a polynucleotide in (a), (b), (c), (d) or (e), above. Thispolynucleotide which hybridizes does not hybridize under stringenthybridization conditions to a polynucleotide having a nucleotidesequence consisting of only A residues or of only T residues. Anadditional nucleic acid embodiment of the invention relates to anisolated nucleic acid molecule comprising a polynucleotide which encodesthe amino acid sequence of an epitope-bearing portion of a BAITpolypeptide having an amino acid sequence in (a), (b), (c) or (d),above.

[0025] The present invention also relates to recombinant vectors, whichinclude the isolated nucleic acid molecules of the present invention,and to host cells containing the recombinant vectors, as well as tomethods of making such vectors and host cells and for using them forproduction of BAIT polypeptides or peptides by recombinant techniques.

[0026] The invention further provides an isolated BAIT polypeptidehaving an amino acid sequence selected from the group consisting of: (a)the amino acid sequence of the BAIT polypeptide having the completeamino acid sequence including the leader sequence shown in FIG. 1 (SEQID NO:2); (b) the amino acid sequence of the mature BAIT polypeptide(without the leader) having the amino acid sequence at positions 19-410in FIG. 1 (SEQ ID NO:2); (c) the amino acid sequence of the BAITpolypeptide having the complete amino acid sequence, including theleader, encoded by the cDNA clone contained in ATCC Deposit No. 97722;and (d) the amino acid sequence of the mature BAIT polypeptide havingthe amino acid sequence encoded by the cDNA clone contained in ATCCDeposit No. 97722. The polypeptides of the present invention alsoinclude polypeptides having an amino acid sequence at least 95%identical, more preferably at least 96% identical, and still morepreferably 97%, 98% or 99% identical to those described in (a), (b), (c)or (d) above, as well as polypeptides having an amino acid sequence withat least 96% similarity, and more preferably at least 97%, 98% or 99%similarity, to those above.

[0027] An additional embodiment of this aspect of the invention relatesto a peptide or polypeptide which has the amino acid sequence of anepitope-bearing portion of a BAIT polypeptide having an amino acidsequence described in (a), (b), (c) or (d), above. Peptides orpolypeptides having the amino acid sequence of an epitope-bearingportion of a BAIT polypeptide of the invention include portions of suchpolypeptides with at least six or seven, preferably at least nine, andmore preferably at least about 30 amino acids to about 50 amino acids,although epitope-bearing polypeptides of any length up to and includingthe complete amino acid sequence of a polypeptide of the inventiondescribed above also are included in the invention.

[0028] In another embodiment, the invention provides an isolatedantibody that binds specifically to a BAIT polypeptide having an aminoacid sequence described in (a), (b), (c) or (d) above. The inventionfurther provides methods for isolating antibodies that bind specificallyto a BAIT polypeptide having an amino acid sequence as described herein.Such antibodies are useful diagnostically or therapeutically asdescribed below.

[0029] The present invention also provides a screening method foridentifying compounds capable of enhancing or inhibiting a biologicalactivity of the BAIT polypeptide, which involves contacting a proteasewhich is inhibited by the BAIT polypeptide with the candidate compoundin the presence of a partially inhibitory amount of BAIT polypeptide,assaying proteolytic activity of the protease on a susceptible substratein the presence of the candidate compound and partially inhibitoryamount of BAIT polypeptide, and comparing the proteolytic activity to astandard level of activity, the standard being assayed when contact ismade between the protease and its substrate in the presence of thepartially inhibitory amount of BAIT polypeptide and the absence of thecandidate compound In this assay, an increase in inhibition ofproteolytic activity over the standard indicates that the candidatecompound is an agonist of BAIT inhibitory activity and a decrease ininhibition of proteolytic activity compared to the standard indicatesthat the compound is an antagonist of BAIT inhibitory activity.

[0030] In another aspect, a screening assay for agonists and antagonistsis provided which involves determining the effect a candidate compoundhas on BAIT binding to the active site of a susceptible protease. Inparticular, the method involves contacting the BAIT-susceptible proteasewith a BAIT polypeptide and a candidate compound and determining whetherBAIT polypeptide binding to the BAIT-susceptible protease is increasedor decreased due to the presence of the candidate compound.

[0031] The present inventor has discovered that BAIT is expressed. inwhole human brain, and to a much lesser extent in adult pancreas andadult heart. For a number of disorders of the central or peripheralnervous system, significantly higher or lower levels of BAIT geneexpression may be detected in certain tissues (e.g., adult brain,embryonic retina, cerebellum and spinal chord) or bodily fluids (e.g.,serum, plasma, urine, synovial fluid or spinal fluid) taken from anindividual having such a disorder, relative to a “standard” BAIT geneexpression level, i.e., the BAIT expression level in healthy tissue froman individual not having the nervous system disorder. Thus, theinvention provides a diagnostic method useful during diagnosis ofnervous system disorders, which involves: (a) assaying BAIT geneexpression level in cells or body fluid of an individual; (b) comparingthe BAIT gene expression level with a standard BAIT gene expressionlevel, whereby an increase or decrease in the assayed BAIT geneexpression level compared to the standard expression level is indicativeof disorder in the nervous system.

[0032] An additional aspect of the invention is related to a method fortreating an individual in need of an increased level of BAIT activity inthe body (i.e., insufficient protease inhibitory activity of BAIT and/orexcessive protease activity of a protease inhabited by BAIT,particularly t-PA), which method comprises administering to such anindividual a composition comprising a therapeutically effective amountof an isolated BAIT polypeptide of the invention or an agonist thereof.Preferred embodiments include a method of treating stoke, braininfarctions, or any other brain disease associated with the loss ofoxygen.

[0033] A still further aspect of the invention is related to a methodfor treating an individual in need of a decreased level of BAIT activityin the body (i.e., less inhibition of a protease susceptible to BAIT)comprising, administering to such an individual a composition comprisinga therapeutically effective amount of a BAIT antagonist. Preferredantagonists for use in the present invention are BAIT-specificantibodies. Preferred embodiments include a method of treating stoke,brain infarctions, or any other brain disease associated with the lossof oxygen.

BRIEF DESCRIPTION OF THE FIGURES

[0034]FIG. 1 shows the nucleotide sequence (SEQ ID NO:1) and deducedamino acid sequence (SEQ ID NO:2) of the human BAIT polypeptide. Theleader sequence of 18 amino acids is underlined.

[0035]FIG. 2 shows the regions of identity between the amino acidsequences of the human BAIT protein and other indicated serpins withwhich the human BAIT polypeptide shares significant homology, asfollows: bovine plasminogen activator inhibitor-1 (BovPAI1; SEQ IDNO:4); rat glial-derived nexin I (RatGDNI; SEQ ID NO:5); mouseantithrombin III (MusATIII; SEQ ID NO:6); chicken neuroserpin (ChkNSP;SEQ ID NO:3). The sequence alignment was generated with the Pileupmodule of the Genetics Computer Group (Wisconsin Package, Version 8,using the parameters GapWeight=3.000, GapLengthWeight=0.100). Thereactive site loops (from positions 415-452 in FIG. 2 (corresponding toBAIT residues 342-378 in FIG. 1; SEQ ID NO:2) are double-underlined, andcritical positions in this sequence are labeled P₁₇ to P₁ and P₁′according to Schechter and Berger, Biochem. Biopys. Res. Commun.27:157-162 (1967). The putative reactive site (cleaved by a targetprotease), between Arg at BAIT position 362 and Met at BAIT position363, is marked with an arrow (

).

[0036]FIG. 3 shows an analysis of the BAIT amino acid sequence. Alpha,beta, turn and coil regions; hydrophilicity and hydrophobicity;amphipathic regions; flexible regions; antigenic index and surfaceprobability are shown. In the “Antigenic Index-Jameson-Wolf” graph, thelocation of the highly antigenic regions of the BAIT protein, i.e.,regions from which epitope-bearing peptides of the invention may beobtained.

[0037]FIG. 4 shows the relationship between the deposited cDNA clone(identified as clone HSDFB 5 50 1×; SEQ ID NO:1) and three related cDNAclones of the invention, designated HPBCT06R (SEQ ID NO:7), HBPDG64R(SEQ ID NO:8), and HPBCR79R (SEQ ID NO:9).

[0038]FIG. 5 shows the results of tests for inhibitory activity ofpurified human BAIT polypeptide on several proteolytic enzymes includingthrombin (2 nM; -Δ-); tissue-type plasminogen activator (tPA, 5 nM;-∘-), urokinase-type plasminogen activator (uPA, 2 nM; -502 -), plasmin(5 nM; -{overscore (V)}-), and trypsin (2 nM; -⋄-).

[0039]FIG. 6 shows rat brain sections 72 hours after reperfusion.Hematoxylin-eosin stain of three representative sections from the samebrain 72 hours after reperfusion. The infarcted area is indicated witharrows, and the box indicates the location where higher resolutionanalysis was performed. Magnification is 5×.

[0040]FIG. 7 shows immunohistochemical staining of BAIT in brain 48hours after reperfusion. Panels A shows the area of penumbra, panel Bshows a similar area of the cortex contralateral to the stroke andpanels C and D show the hippocampus. Panels A and C are ipsilateral tothe stroke and panels B and D are contralateral. Magnification is 100×in A and B and 40× in C and D.

[0041]FIG. 8 shows quantitative analysis of infarct volume 72 hoursafter reperfusion. Quantitation of the stroke volume was performed asdescribed in the Examples. PBS: animals injected with PBS (n=8); Ns:animals injected with BAIT (n=8): Cl-Ns: animals injected withelastase-cleaved inactive BAIT (n=2). P values relative to thePBS-treated animals <0.01 are shown, and errors represent S.E.M.

[0042]FIG. 9 shows SDS-PAGE zymography of brain extracts. Panel A isSDS-PAGE zymography of brain extracts. Lane 1 is human tPA, lane 2 is arat kidney extract as a marker for rat uPA, lanes 3-6 are extracts ofbrain 6 hours after reperfusion and lanes 7-10 are extracts 72 hoursafter reperfusion. Lanes 3 and 7 are ipsilateral to the infarct of PBStreated animals, lanes 4 and 8 are contralateral to the infarct. Lanes 5and 9 are ipsilateral to the infarct in BAIT treated animals and lanes 6and 10 are contralateral. Panel B shows quantitative image analysis ofPA activity from SDS-PAGE zymography of brain extracts 6 hours followingreperfusion. The results represent the average fold increase in eithertPA or uPA activity ipsilateral to the stroke relative to normalbaseline PA activities contralateral to the stroke. PBS and Ns representanimals treated with either PBS or BAIT respectively, and n≧3 for eachcondition tested. P values ≦0.05 relative to the contralateral activityare shown, and errors represent S.E.M.

[0043]FIG. 10 shows in situ zymography and immununohistochemicalstaining of brain sections. In situ zymography in panels A-F; panels Aand D are developed without plasminogen, and all other panels aredeveloped with plasminogen. Panels C and F also contain anti-tPAantibodies. The white arrows indicate the area of the infarct.Magnification is 3×. Panel G is immunohistochemical staining of tPA 6hours after reperfusion. The black arrows indicate tPA positive bloodvessels. Panel H is immunohistochemical staining of uPA in the area ofpenumbra 72 hours after reperfusion. The magnification in panels G and His 400×.

[0044]FIG. 11 shows immunohistochemical staining of laminin. Panels Aand B show normal cortex in an animal without stroke. Panel A wasdeveloped with anti- laminin but without pretreatment in vitro of thesection with proteinase. Panel B is an adjacent section with in vitroproteinase treatment. Panels C-F are from stroked animals and developedwith anti-laminin and no proteinase treatment. Panels C-D are 10 minutesafter reperfusion and panels E-F are 6 hours after reperfusion. Panels Cand E represent PBS-treated and panels D and F BAIT-treated animals.Magnification is 100×.

[0045]FIG. 12 shows neuronal apoptosis within the ischemic penumbra.TUNEL staining ipsilateral of the infarct in PBS-treated (panels A & B)and BAIT-treated (panel C) animals. In panel A, NC indicates thenecrotic core, and P indicates the area of the penumbra. Panels B-C arehigh magnification images of the penumbra in control (panel B) and BAITtreated animals (panel C). Examples of cells considered to be apoptoticfor the purposes of quantification are indicated with the open arrowswhile cells considered as necrotic are indicated with the closed arrows.Magnification in panel A is 40× and in panels B-C 400×. Panel D,quantitative analysis of apoptosis in the area of penumbra 72 hoursafter reperfusion. Quantitation was performed as described in theExamples and only cells with apoptotic bodies present (see panel B) werecounted. Control represents animals injected with PBS (n=6). BAITindicates animals injected with BAIT (n=6). P values <0.05 are shown,and errors represent S.E.M. Panel E, quantitation of apoptosis wasperformed as in panel D at the times indicated, (ο) PBS treated animals,(□) BAIT treated.

DETAILED DESCRIPTION

[0046] The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding a human BAIT polypeptide having thea-amino acid sequence shown in FIG. 1 (SEQ ID NO:2), which wasdetermined by sequencing a cloned cDNA. The nucleotide sequence shown inFIG. 1 (SEQ ID NO:1) was obtained by sequencing the HSDFB55SO1 clone,which was deposited on September 18, 1996 at the American Type CultureCollection, 10801 University Boulevard, Manassas, Va. 20110, and givenaccession number ATCC 97722. The deposited clone is contained in thepBluescript SK(−) plasmid (Stratagene, La Jolla, Calif.).

[0047] Nucleic Acid Molecules

[0048] Unless otherwise indicated, all nucleotide sequences determinedby sequencing a DNA molecule herein were determined using an automatedDNA sequencer (such as the Model 373 from Applied Biosystems, Inc.,Foster City, Calif.), and all amino acid sequences of polypeptidesencoded by DNA molecules determined herein were predicted by translationof a DNA sequence determined as above. Therefore, as is known in the artfor any DNA sequence determined as above approach, any nucleotidesequence determined herein may contain some errors. Nucleotide sequencesdetermined by automation are typically at least about 99% identical,more typically at least about 99.1% to at least about 99.9% identical tothe actual nucleotide sequence of the sequenced DNA molecule. The actualsequence can be more precisely determined by other approaches includingmanual DNA sequencing methods well known in the art. As is also known inthe art, a single insertion or deletion in a determined nucleotidesequence compared to the actual sequence will cause a frame shift intranslation of the nucleotide sequence such that the predicted aminoacid sequence encoded by a determined nucleotide sequence will becompletely different from the amino acid sequence actually encoded bythe sequenced DNA molecule, beginning at the point of such an insertionor deletion.

[0049] Unless otherwise indicated, each “nucleotide sequence” set forthherein is presented as a sequence of deoxyribonucleotides (abbreviatedA, G, C and T). However, by “nucleotide sequence” of a nucleic acidmolecule or polynucleotide is intended, for a DNA molecule orpolynucleotide, a sequence of deoxyribonucleotides, and for an RNAmolecule or polynucleotide, the corresponding sequence ofribonucleotides (A, G, C and U), where each thymidinedeoxyribonucleotide (T) in the specified deoxyribonucleotide sequence isreplaced by the ribonucleotide uridine (U). For instance, reference toan RNA molecule having the sequence of SEQ ID NO:1 set forth usingdeoxyribonucleotide abbreviations is intended to indicate an RNAmolecule having a sequence in which each deoxyribonucleotide A, G or Cof SEQ ID NO:1 has been replaced by the corresponding ribonucleotide A,G or C, and each deoxyribonucleotide T has been replaced by aribonucleotide U. Using the information provided herein, such as thenucleotide sequence in FIG. 1, a nucleic acid molecule of the presentinvention encoding a BAIT polypeptide may be obtained using standardcloning and screening procedures, such as those for cloning cDNAs usingmRNA as starting material. Illustrative of the invention, the nucleicacid molecule described in FIG. 1 (SEQ ID NO:1) was discovered in a cDNAlibrary derived from whole human brain. Additional cDNA clones of theBAIT gene were also identified in cDNA libraries from the followingtissues: spinal cord, pineal gland and adrenal gland tumor. Thedetermined nucleotide sequence of the BAIT cDNA of FIG. 1 (SEQ ID NO:1)contains an open reading frame encoding a protein of 410 amino acidresidues, with an initiation codon at positions 89-91, and a predictedmolecular weight of about 46.4 kDa. The encoded polypeptide has a leadersequence of 18 amino acids, underlined in FIG. 1; and the amino acidsequence of the expressed mature protein is also shown in FIG. 1, asamino acid residues 19-410 (SEQ ID NO:2). The amino acid sequence of theBAIT protein shown in FIG. 1 (SEQ ID NO:2) is about 80% identical to thepublished mRNA for chicken neuroserpin (Osterwalder, T., et al., 1996,supra) as shown in FIG. 2. FIG. 2 shows the regions of identity betweenthe amino acid sequences of the human BAIT protein and other indicatedserpins with which the human BAIT polypeptide shares significanthomology, as follows: bovine plasminogen activator inhibitor-1 (BovPAI1;SEQ ID NO:4); rat glial-derived nexin I RatGDNI; SEQ ID NO:5); mouseantithrombin III (MusATIII SEQ ID NO:6); chicken neuroserpin (ChkNSP;SEQ ID NO:3).

[0050] Sequence comparisons suggest that the chicken neuroserpin andBAIT are orthologs of one another and are distantly related to thebetter characterized mammalian serpins seen in FIG. 2. There is 77%homology at the DNA level between BAIT and neuroserpin which translatesinto 90% and 80% amino acid similarity and identity, respectively. Aminoacid identities between the non-human mammalian serpins and BAIT drop toabout 30%. Moreover, within the functionally important reactive siteloop, there is only one conservative amino acid change between BAIT andneuroserpin. There are 7 non-conservative changes between BAIT and PAI-1in the same 38 amino acid region. The active site P1-P1′ residues,however, are perfectly conserved between BAIT, neuroserpin, and PAI-1.The BAIT region corresponding to the ATIII heparin-binding site has 4acidic amino acids which implies that heparin is not a co-factor as itis with ATIII. One potentially significant difference between BAIT andneuroserpin is the presence of 3 consensus N-linked glycosylation sitesin the former versus 2 in the latter. Thus, BAIT and neuroserpin arelikely to have similar enzymatic properties which may not overlap thoseof the related serpins.

[0051] Leader and Mature Sequences

[0052] The amino acid sequence of the complete BAIT protein includes aleader sequence and a mature protein, as shown in FIG. 1 (SEQ ID NO:2).More in particular, the present invention provides nucleic acidmolecules encoding one or more mature form(s) of the BAIT protein. Thus,according to the signal hypothesis, proteins secreted by mammalian cellshave a signal or secretory leader sequence which is cleaved from themature protein once export of the growing protein chain across the roughendoplasmic reticulum has been initiated. Most mammalian cells and eveninsect cells cleave secreted proteins with the same specificity.However, in some cases, cleavage of a secreted protein is not entirelyuniform, which results in two or more mature species of the protein.Further, it has long been known that the cleavage specificity of asecreted protein is ultimately determined by the primary structure ofthe complete protein, that is, it is inherent in the amino acid sequenceof the polypeptide. Therefore, the present invention provides anucleotide sequence encoding the mature BAIT polypeptide having theamino acid sequence encoded by the cDNA clone contained in the hostidentified as ATCC Deposit No. 97722. By the “mature BAIT polypeptidehaving the amino acid sequence encoded by the cDNA clone in ATCC DepositNo. 97722” is meant the mature form(s) of the BAIT protein produced byexpression in a mammalian cell (e.g., COS cells, as described below) ofthe complete open reading frame encoded by the human DNA sequence of theclone contained in the vector in the deposited host.

[0053] In the present case, the deposited cDNA has been expressed ininsect cells using a baculovirus expression vector, as describedhereinbelow; and amino acid sequencing of the amino terminus of thesecreted species indicated that the N-terminus of the mature BAITprotein comprises the amino acid sequence beginning at amino acid 19 ofFIG. 1 (SEQ ID NO:2). Thus, the leader sequence of the BAIT protein inthe amino acid sequence of FIG. 1 is 18 amino acids, from position 1 to18 in FIG. 1 (SEQ ID NO:2).

[0054] The predicted 410 amino acids of the complete BAIT (prepro)polypeptide is expected to yield a 46.4 kDa band. The observed doubletband of 45 and 46 kDa upon expression in the baculovirus system waswithin the expected size range when the putative 18 amino acid signalpeptide is removed. The approximate 1 kDa difference in the observeddoublet bands may be explained by differential glycosylation. Evidenceto support this includes the three consensus N-linked glycosylation sitepresent in the nucleotide sequence (FIG. 1) and the presence ofoligosaccharide moieties on the purified protein determinedexperimentally.

[0055] N-Terminal and C-Terminal Deletion Mutants

[0056] In addition to the mature form of a protein being biologicallyactive, it is known in the art for many proteins, including the matureform(s) of a secreted protein, that one or more amino acids may bedeleted from the N-terminus without substantial loss of biologicalfunction. In the present case, deletions of at least up to 30 N-terminalamino acids from the end of the mature (secreted) polypeptide may retainsome biological activity such as binding to the active site of at leastone protease. However, even if deletion of one or more amino acids fromthe N-terminus of a protein results in modification of loss of one ormore biological functions of the protein, other biological activitiesmay still be retained. Thus, the ability of the shortened protein toinduce and/or binding to antibodies which recognize the complete ormature protein generally will be retained when less than the majority ofthe residues of the complete or mature protein are removed from theN-terminus. Whether a particular polypeptide lacking N-terminalresidues, of a complete protein retains such immunologic activities canreadily be determined by routine methods described herein and otherwiseknown in the art. Similarly, deletion of one or more amino acids fromthe C-terminus of a protein also may provide shortened polypeptideswhich retain some or all biological activities.

[0057] In the baculovirus expression system the BAIT polypeptide wasprocessed to produce multiple forms of BAIT. Beginning after the 18amino acid leader, the next amino acids found on the baculovirusprocessed BAIT are as follows:

[0058] ATFPE: 40%

[0059] TFPEE: 30%

[0060] MPEEA: 10%

[0061] These are found within the first 7 amino acids of the mature BAITin FIG. 1. Therefor, there are multiple different N-terminal amino acidson the BAIT produced in the Baculovirus system.

[0062] Accordingly, the present invention further provides polypeptideshaving one or more residues from the amino terminus of the amino acidsequence of the complete BAIT polypeptide in SEQ ID NO:2, up to 30residues from the amino terminus after the leader cleavage sitedescribed above, and polynucleotides encoding such polypeptides. Inparticular, the present invention provides polypeptides having the aminoacid sequence of residues n-410 of the amino acid sequence in SEQ IDNO:2, where n is any integer in the range of 2-410, and preferably inthe range of 2-49 specified range and 49 is the position of the 30thresidue from the N-terminus of the mature polypeptide, after the aboveleader cleavage site, as shown in the amino acid sequence in SEQ IDNO:2. More in particular, the invention provides polypeptides having theamino acid sequence of residues 2-410, 3-410, 4-410, 5-410, 6-410,7-410, 8-410, 9-410, 10-410, 11-410, 12-410, 13-410, 14-410, 15-410,16-410, 17-410, 18-410, 19-410, 20-410, 21-410, 22-410, 23-410, 24-410,25-410, 26-410, 27-410, 28-410, 29-410, 30-410, 31-410, 32-410, 33-410,34-410, 35-410, 36-410, 37-410, 38-410, 39-410, 40-410, 41-410, 42-410,43-410, 44-410, 45-410, 46-410, 47-410, 48-410 and 49-410 of SEQ IDNO:2. Polynucleotides encoding these polypeptides also are provided.

[0063] More in particular, the invention provides polynucleotidesencoding polypeptides comprising, or alternatively consisting of, theamino acid sequence of residues of: A-2 to L-410; F-3 to L-410; L-4 toL-410; G-5 to L-410; L-6 to L-410; F-7 to L-410; S-8 to L-410; L-9 toL-410; L-10 to L-410; V-11 to L-410; L-12 to L-410; Q-13 to L-410; S-14to L-410; M-15 to L-410; A-16 to L-410; T-17 to L-410; G-18 to L-410;A-19 to L-410; T-20 to L-410; F-21 to L-410; P-22 to L-410; E-23 toL-410; E-24 to L-410; A-25 to L-410; I-26 to L-410; A-27 to L-410; D-28to L-410; L-29 to L-410; S-30 to L-410; V-31 to L-410; N-32 to L-410;M-33 to L-410; Y-34 to L-410; N-35 to L-410; R-36 to L-410; L-37 toL-410; R-38 to L-410; A-39 to L-410; T-40 to L-410; G-41 to L-410; E-42to L-410; D-43 to L-410; E-44 to L-410; N-45 to L-410; I-46 to L-410;L-47 to L-410; F-48 to L-410; S-49 to L-410; P-50 to L-410; L-51 toL-410; S-52 to L-410; 1-53 to L-410; A-54 to L-410; L-55 to L-410; A-56to L-410; M-57 to L-410; G-58 to L-410; M-59 to L-410; M-60 to L-410;E-61 to L-410; L-62 to L-410; G-63 to L-410; A-64 to L-410; Q-65 toL-410; G-66 to L-410; S-67 to L-410; T-68 to L-410; Q-69 to L-410; K-70to L-410; E-71 to L-410; 1-72 to L-410; R-73 to L-410; H-74 to L-410;S-75 to L-410; M-76 to L-410; G-77 to L-410; Y-78 to L-410; D-79 toL-410; S-80 to L-410; L-81 to L-410; K-82 to L-410; N-83 to L-410; G-84to L-410; E-85 to L-410; E-86 to L-410; F-87 to L-410; S-88 to L-410;F-89 to L-410; L-90 to L-410; K-91 to L-410; E-92 to L-410; F-93 toL-410; S-94 to L-410; N-95 to L-410; M-96 to L-410; V-97 to L-410; T-98to L-410; A-99 to L-410; K-100 to L-410; E-101 to L-410; S-102 to L-410;Q-103 to L-410; Y-104 to L-410; V-105 to L-410; M-106 to L-410; K-107 toL-410; 1-108 to L-410; A-109 to L-410; N-110 to L-410; S-111 to L- 410;L-112 to L-410; F-113 to L-410; V-114 to L-410; Q-115 to L-410; N-116 toL-410; G-117 to L-410; F-118 to L-410; H-119 to L-410; V-120 to L-410;N-121 to L-410; E-122 to L-410; E-123 to L-410; F-124 to L-410; L-125 toL-410; Q-126 to L-410; M-127 to L-410; M-128 to L-410; K-129 to L-410;K-130 to L-410; Y-131 to L-410; F-132 to L-410; N-133 to L-410; A-134 toL-410; A-135 to L-410; V-136 to L-410; N-137 to L-410; H-138 to L-410;V-139 to L-410; D-140 to L-410; F-141 to L-410; S-142 to L-410; Q-143 toL-410; N-144 to L-410; V-145 to L-410; A-146 to L-410; V-147 to L-410;A-148 to L-410; N-149 to L-410; Y-150 to L-410; 1-151 to L-410; N-152 toL-410; K-153 to L-410; W-154 to L-410; V-155 to L-410; E-156 to L-410;N-157 to L-410; N-158 to L-410; T-159 to L-410; N-160 to L-410; N-161 toL-410; L-162 to L-410; V-163 to L-410; K-164 to L-410; D-165 to L-410;L-166 to L-410; V-167 to L-410; S-168 to L-410; P-169 to L-410; R-170 toL-410; D-171 to L-410; F-172 to L-410; D-173 to L-410; A-174 to L-410;A-175 to L-410; T-176 to L-410; Y-177 to L-410; L-178 to L-410; A-179 toL-410; L-180 to L-410; 1-181 to L-410; N-182 to L-410; A-183 to L-410;V-184 to L-410; Y-185 to L-410; F-186 to L-410; K-187 to L-410; G-188 toL-410; N-189 to L-410; W-190 to L-410; K-191 to L-410; S-192 to L-410;Q-193 to L-410; F-194 to L-410; R-195 to L-410; P-196 to L-410; E-197 toL-410; N-198 to L-410; T-199 to L-410; R-200 to L-410; T-201 to L-410;F-202 to L-410; S-203 to L-410; F-204 to L-410; T-205 to L-410; K-206 toL-410; D-207 to L-410; D-208 to L-410; E-209 to L-410; S-210 to L-410;E-211 to L-410; V-212 to L-410; Q-213 to L-410; 1-214 to L-410; P-215 toL-410; M-216 to L-410; M-217 to L-410; Y-218 to L-410; Q-219 to L-410;Q-220 to L-410; G-221 to L-410; E-222 to L-410; F-223 to L-410; Y-224 toL-410; Y-225 to L-410; G-226 to L-410; E-227 to L-410; F-228 to L-410;S-229 to L-410; D-230 to L-410; G-231 to L-410; S-232 to L-410; N-233 toL-410; E-234 to L-410; A-235 to L-410; G-236 to L-410; G-237 to L-410;1-238 to L-410; Y-239 to L-410; Q-240 to L-410; V-241 to L-410; L-242 toL-410; E-243 to L-410; I-244 to L-410; P-245 to L-410; Y-246 to L-410;E-247 to L-410; G-248 to L-410; D-249 to L-410; E-250 to L-410; 1-251 toL-410; S-252 to L-410; M-253 to L-410; M-254 to L-410; L-255 to L-410;V-256 to L-410; L-257 to L-410; S-258 to L-410; R-259 to L-410; Q-260 toL-410; E-261 to L-410; V-262 to L-410; P-263 to L-410; L-264 to L-410;A-265 to L-410; T-266 to L-410; L-267 to L-410; E-268 to L-410; P-269 toL-410; L-270 to L-410; V-271 to L-410; K-272 to L-410; A-273 to L-410;Q-274 to L-410; L-275 to L-410; V-276 to L-410; E-277 to L-410; E-278 toL-410; W-279 to L-410; A-280 to L-410; N-281 to L-410; S-282 to L-410;V-283 to L-410; K-284 to L-410; K-285 to L-410; Q-286 to L-410; K-287 toL-410; V-288 to L-410; E-289 to L-410; V-290 to L-410; Y-291 to L-410;L-292 to L-410; P-293 to L-410; R-294 to L-410; F-295 to L-410; T-296 toL-410; V-297 to L-410; E-298 to L-410; Q-299 to L-410; E-300 to L-410;1-301 to L-410; D-302 to L-410; L-303 to L-410; K-304 to L-410; D-305 toL-410; V-306 to L-410; L-307 to L-410; K-308 to L-410; A-309 to L-410;L-310 to L-410; G-311 to L-410; 1-312 to L-410; T-313 to L-410; E-314 toL-410; L-315 to L-410; F-36 to L-410;-317 to L-410; K-318 to L-410;D-319 to L-410; A -32 0 to L-410; N -32 1 to L-410; L-32 2 to L-410;T-323 to L-410; G-32 4 to L-410; L-325 to L-410; S-326 to L-410; D-327to L-410; N-328 to L-410; K-329 to L-410; E-330 to L-410; I-331 toL-410; F-332 to L-410; L-333 to L-410; S-334 to L-410; K-335 to L-410;A-336 to L-410; 1-337 to L-410; H-338 to L-410; K-339 to L-410; S-340 toL-410; F -341 to L-410; L-342 to L-410; E-343 to L-410; V-344 to L-410;N-345 to L-410; E-346 to L-410; E-347 to L-410; G-348 to L-410; S-349 toL-410; E-350 to L-410; A-351 to L-410; A-352 to L-410; A-353 to L-410;V-354 to L-410; S-355 to L-410; G-356 to L-410; M-357 to L-410; I-358 toL-410; A-359 to L-410; I-360 to L-410; S-361 to L-410; R-362 to L-410;M-363 to L-410; A-364 to L-410; V-365 to L-410; L-366 to L-410; Y-367 toL-410; P-368 to L-410; Q-369 to L-410; V-370 to L-410; I-371 to L-410;V-372 to L-410; D-373 to L-410; H-374 to L-410; P-375 to L-410; F-376 toL-410; F-377 to L-410; F-378 to L-410; L-379 to L-410; 1-380 to L-410;R-381 to L-410; N-382 to L-410; R-383 to L-410; R-384 to L-410; T-385 toL-410; G-386 to L-410; T-387 to L-410; 1-388 to L-410; L-389 to L-410;F-390 to L-410; M-391 to L-410; G-392 to L-410; R-393 to L-410; V-394 toL-410; M-395 to L-410; H-396 to L-410; P-397 to L-410; E-398 to L-410;T-399 to L-410; M-400 to L-410; N-401 to L-410; T-402 to L-410; S-403 toL-410; G-404 to L-410; and/or H-405 to L-410 of SEQ ID NO:2.Polynucleotides encoding these polypeptides are also encompassed by theinvention.

[0064] The present application is also directed to nucleic acidmolecules comprising, or alternatively, consisting of, a polynucleotidesequence at least 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to thepolynucleotide sequence encoding the BAIT polypeptide described above.The present invention also encompasses the above polynucleotidesequences fused to a heterologous polynucleotide sequence.

[0065] Similarly, the present invention further provides polypeptideshaving one or more residues from the carboxyl terminus of the amino acidsequence of the complete BAIT polypeptide in SEQ ID NO:2, up to 30residues from the carboxyl terminus, and polynucleotides encoding suchpolypeptides. In particular, the present invention provides polypeptideshaving the amino acid sequence of residues 1-m of the amino acidsequence in SEQ ID NO:2, where m is any integer in the range of 2-410,and preferably in the range of 381-409, as shown in the amino acidsequence in SEQ ID NO:2. More in particular, the invention providespolypeptides having the amino acid sequence of residues 1-381, 1-382,1-383, 1-384, 1-385, 1-386, 1-387, etc. up to 1-408 of SEQ ID NO:2.Polynucleotides encoding these polypeptides also are provided. Inaddition, polypeptides (and polynucleotides encoding these) having bothN-terminal and C-terminal deletions together, of the general formula n-mof SEQ ID NO:2 are included, where n and m are integers as definedabove.

[0066] Even if deletion of one or more amino acids from the N-terminusand/or the C-terminus of a protein results in modification of loss ofone or more biological functions of the protein, other functionalactivities (e.g., biological activities, ability to multimerize, abilityto bind BAIT ligand) may still be retained. For example the ability ofthe shortened BAIT mutein to induce and/or bind to antibodies whichrecognize the complete or mature forms of the polypeptide generally willbe retained when less than the majority of the residues of the completeor mature polypeptide are removed from the N-terminus and/or theC-terminus. Whether a particular polypeptide lacking N-terminus and/orC-terminal residues of a complete polypeptide retains such immunologicactivities can readily be determined by routine methods described hereinand otherwise known in the art. It is not unlikely that an BAIT muteinwith a large number of deleted N-terminal and/or C-terminal amino acidresidues may retain some biological or immunogenic activities. In fact,peptides composed of as few as six BAIT amino acid residues may oftenevoke an immune response.

[0067] More in particular, the invention provides polynucleotidesencoding polypeptides comprising, or alternatively consisting of, theamino acid sequence of residues of: P-22 to E-409; P-22 to E-408; P-22to F-407; P-22 to D-406; P-22 to H-405; P-22 to G-404; P-22 to S-403;P-22 to T-402; P-22 to N-401; P-22 to M-400; P-22 to T-399; P-22 toE-398; P-22 to P-397; P-22 to H-396; P-22 to M-395; P-22 to V-394; P-22to R-393; P-22 to G-392; P-22 to M-391; P-22 to F-390; P-22 to L-389;P-22 to I-388; P-22 to T-387; P-22 to G-386; P-22 to T-385; P-22 toR-384; P-22 to R-383; P-22 to N-382; P-22 to R-381; P-22 to I-380; P-22to L-379; P-22 to F-378; P-22 to F-377; P-22 to F-376; P-22 to P-375;P-22 to H-374; P-22 to D-373; P-22 to V-372; P-22 to I-371; P-22 toV-370; P-22 to Q-369; P-22 to P-368; P-22 to Y-367; P-22 to L-366; P-22to V-365; P-22 to A-364; P-22 to M-363; P-22 to R-362; P-22 to S-361;P-22 to I-360; P-22 to A-359; P-22 to I-358; P-22 to M-357; P-22 toG-356; P-22 to S-355; P-22 to V-354; P-22 to A-353; P-22 to A-352; P-22to A-351; P-22 to E-350; P-22 to S-349; P-22 to G-348; P-22 to E-347;P-22 to E-346; P-22 to N-345; P-22 to V-344; P-22 to E-343; P-22 toL-342; P-22 to F-341; P-22 to S-340; P-22 to K-339; P-22 to H-338; P-22to I-337; P-22 to A-336; P-22 to K-335; P-22 to S-334; P-22 to L-333;P-22 to F-332; P-22 to I-331; P-22 to E-330; P-22 to K-329; P-22 toN-328; P-22 to D-327; P-22 to S-326; P-22 to L-325; P-22 to G-324; P-22to T-323; P-22 to L-322; P-22 to N-321; P-22 to A-320; P-22 to D-319;P-22 to K-318; P-22 to I-317; P-22 to F-316; P-22 to I-315; P-22 toE-314; P-22 to T-313; P-22 to I-312; P-22 to G-311; P-22 to L-310; P-22to A-309; P-22 to K-308; P-22 to L-307; P-22 to V-306; P-22 to D-305;P-22 to K-304; P-22 to L-303; P-22 to D-302; P-22 to I-301; P-22 toE-300; P-22 to Q-299; P-22 to E-298; P-22 to V-297; P-22 to T-296; P-22to F-295; P-22 to R-294; P-22 to P-293; P-22 to L-292; P-22 to Y-291;P-22 to V-290; P-22 to E-289; P-22 to V-288; P-22 to K-287; P-22 toQ-286; P-22 to K-285; P-22 to K-284; P-22 to V-283; P-22 to S-282; P-22to N-281; P-22 to A-280; P-22 to W-279; P-22 to E-278; P-22 to E-277;P-22 to V-276; P-22 to L-275; P-22 to Q-274; P-22 to A-273; P-22 toK-272; P-22 to V-271; P-22 to L-270; P-22 to P-269; P-22 to E-268; P-22to L-267; P-22 to T-266; P-22 to A-265; P-22 to L-264; P-22 to P-263;P-22 to V-262; P-22 to E-261; P-22 to Q-260; P-22 to R-259; P-22 toS-258; P-22 to L-257; P-22 to V-256; P-22 to L-255; P-22 to M-254; P-22to M-253; P-22 to S-252; P-22 to I-251; P-22 to E-250; P-22 to D-249;P-22 to G-248; P-22 to E-247; P-22 to Y-246; P-22 to P-245; P-22 toI-244; P-22 to E-243; P-22 to L-242; P-22 to V-241; P-22 to Q-240; P-22to Y-239; P-22 to I-238; P-22 to G-237; P-22 to G-236; P-22 to A-235;P-22 to E-234; P-22 to N-233; P-22 to S-232; P-22 to G-231; P-22 toD-230; P-22 to S-229; P-22 to F-228; P-22 to E-227; P-22 to G-226; P-22to Y-225; P-22 to Y-224; P-22 to F-223; P-22 to E-222; P-22 to G-221;P-22 to Q-220; P-22 to Q-219; P-22 to Y-218; P-22 to M-217; P-22 toM-216; P-22 to P-215; P-22 to I-214; P-22 to Q-213; P-22 to V-212; P-22to E-211; P-22 to S-210; P-22 to E-209; P-22 to D-208; P-22 to D-207;P-22 to K-206; P-22 to T-205; P-22 to F-204; P-22 to S-203; P-22 toF-202; P-22 to T-201; P-22 to R-200; P-22 to T-199; P-22 to N-198; P-22to E-197; P-22 to P-196; P-22 to R-195; P-22 to F-194; P-22 to Q-193;P-22 to S-192; P-22 to K-191; P-22 to W-190; P-22 to N-189; P-22 toG-188; P-22 to K-187; P-22 to F-186; P-22 to Y-185; P-22 to V-184; P-22to A-183; P-22 to N-182; P-22 to I-181; P-22 to L-180; P-22 to A-179;P-22 to L-178; P-22 to Y-177; P-22 to T-176; P-22 to A-175; P-22 toA-174; P-22 to D-173; P-22 to F-172; P-22 to D-171; P-22 to R-170; P-22to P-169; P-22 to S-168; P-22 to V-167; P-22 to L-166; P-22 to D-165;P-22 to K-164; P-22 to V-163; P-22 to L-162; P-22 to N-161; P-22 toN-160; P-22 to T-159; P-22 to N-158; P-22 to N-157; P-22 to E-156; P-22to V-155; P-22 to W-154; P-22 to K-153; P-22 to N-152; P-22 to I-151;P-22 to Y-150; P-22 to N-149; P-22 to A-148; P-22 to V-147; P-22 toA-146; P-22 to V-145; P-22 to N-144; P-22 to Q-143; P-22 to S-142; P-22to F-141; P-22 to D-140; P-22 to V-139; P-22 to H-138; P-22 to N-137;P-22 to V-136; P-22 to A-135; P-22 to A-134; P-22 to N-133; P-22 toF-132; P-22 to Y-131; P-22 to K-130; P-22 to K-129; P-22 to M-128; P-22to M-127; P-22 to Q-126; P-22 to L-125; P-22 to F-124; P-22 to E-123;P-22 to E-122; P-22 to N-121; P-22 to V-120; P-22 to H-1 19; P-22 toF-118; P-22 to G-117; P-22 to N-1 16; P-22 to Q-115; P-22 to V-114; P-22to F-113; P-22 to L-112; P-22 to S-11; P-22 to N-110; P-22 to A-109;P-22 to I-108; P-22 to K-107; P-22 to M-106; P-22 to V-105; P-22 toY-104; P-22 to Q-103; P-22 to S-102; P-22 to E-101; P-22 to K-100; P-22to A-99; P-22 to T-98; P-22 to V-97; P-22 to M-96; P-22 to N-95; P-22 toS-94; P-22 to F-93; P-22 to E-92; P-22 to K-9 1; P-22 to L-90; P-22 toF-89; P-22 to S-88; P-22 to F-87; P-22 to E-86; P-22 to E-85; P-22 toG-84; P-22 to N-83; P-22 to K-82; P-22 to L-81; P-22 to S-80; P-22 toD-79; P-22 to Y-78; P-22 to G-77; P-22 to M-76; P-22 to S-75; P-22 toH-74; P-22 to R-73; P-22 to I-72; P-22 to E-71; P-22 to K-70; P-22 toQ-69; P-22 to T-68; P-22 to S-67; P-22 to G-66; P-22 to Q-65; P-22 toA-64; P-22 to G-63; P-22 to L-62; P-22 to E-61; P-22 to M-60; P-22 to M-59; P-22 to G-58; P-22 to M-57; P-22 to A-56; P-22 to L-55; P-22 toA-54; P-22 to I-53; P-22 to S-52; P-22 to L-51; P-22 to P-50; P-22 toS-49; P-22 to F-48; P-22 to L-47; P-22 to I-46; P-22 to N-45; P-22 toE-44; P-22 to D-43; P-22 to E-42; P-22 to G-41; P-22 to T-40; P-22 toA-39; P-22 to R-38; P-22 to L-37; P-22 to R-36; P-22 to N-35; P-22 toY-34; P-22 to M-33; P-22 to N-32; P-22 to V-31; P-22 to S-30; P-22 toL-29; and/or P-22 to D-28 of SEQ ID NO:2. Polynucleotides encoding thesepolypeptides are also encompassed by the invention.

[0068] Moreover, a signal sequence may be added to these C-terminalcontructs. For example, amino acids 1-21 of SEQ ID NO:2, amino acids2-21 of SEQ ID NO:2, amino acids 3-21 of SEQ ID NO:2, amino acids 4-21of SEQ ID NO:2, amino acids 5-21 of SEQ ID NO:2, amino acids 6-21 of SEQID NO:2, amino acids 7-21 of SEQ ID NO:2, amino acids 8-21 of SEQ IDNO:2, amino acids 9-21 of SEQ ID NO:2, amino acids 10-21 of SEQ ID NO:2,amino acids 11-21 of SEQ ID NO:2, amino acids 12-21 of SEQ ID NO:2,amino acids 13-21 of SEQ ID NO:2, amino acids 14-21 of SEQ ID NO:2,amino acids 15-21 of SEQ ID NO:2, amino acids 16-21 of SEQ ID NO:2,amino acids 17-21 of SEQ ID NO:2, amino acids 18-21 of SEQ ID NO:2,amino acids 19-21 of SEQ ID NO:2, amino acids 20-21 of SEQ ID NO:2,and/or amino acids 21 of SEQ ID NO:2 can be added to the N-terminus ofeach C-terminal constructs listed above. Additionally, a methioine canbe added to these constructs.

[0069] The present application is also directed to nucleic acidmolecules comprising, or alternatively, consisting of, a polynucleotidesequence at least 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to thepolynucleotide sequence encoding the BAIT polypeptide described above.The present invention also encompasses the above polynucleotidesequences fused to a heterologous polynucleotide sequence. In addition,any of the above listed N- or C-terminal deletions can be combined toproduce a N- and C-terminal deleted BAIT polypeptide. The invention alsoprovides polypeptides having one or more amino acids deleted from boththe amino and the carboxyl termini, which may be described generally ashaving residues m-n of SEQ ID NO:2, where n and m are integers asdescribed above. Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

[0070] Also included are a nucleotide sequence encoding a polypeptideconsisting of a portion of the complete BAIT amino acid sequence encodedby the cDNA clone contained in ATCC Deposit No.97722, where this portionexcludes any integer of amino acid residues from 1 to about 400 aminoacids from the amino terminus of the complete amino acid sequenceencoded by the cDNA clone contained in ATCC Deposit No. 97722, or anyinteger of amino acid residues from 1 to about 400 amino acids from thecarboxy terminus, or any combination of the above amino terminal andcarboxy terminal deletions, of the complete amino acid sequence encodedby the cDNA clone contained in ATCC Deposit No. 97722. Polynucleotidesencoding all of the above deletion mutant polypeptide forms also areprovided.

[0071] The present application is also directed to proteins containingpolypeptides at least 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to the polypeptide sequence set forth herein m-n. In preferredembodiments, the application is directed to proteins containingpolypeptides at least 90%, 95%, 96%, 97%, 98% or 99% identical topolypeptides having the amino acid sequence of the specific BAIT N- andC-terminal deletions recited herein. Polynucleotides encoding thesepolypeptides are also encompassed by the invention.

[0072] As indicated, nucleic acid molecules of the present invention maybe in the form of RNA, such as mRNA, are in the form of DNA, including,for instance, cDNA and genomic DNA obtained by cloning or producedsynthetically. The DNA may be double-stranded or single-stranded.Single-stranded DNA or RNA may be the coding strand, also known as thesense strand, or it may be the non-coding strand, also referred to asthe anti-sense strand.

[0073] By “isolated” nucleic acid molecule(s) is intended a nucleic acidmolecule, DNA or RNA, which has been removed from its native environment. For example, recombinant DNA molecules contained in a vector areconsidered isolated for the purposes of the present invention. Furtherexamples of isolated DNA molecules include recombinant DNA moleculesmaintained in heterologous host cells or purified (partially orsubstantially) DNA molecules in solution. Isolated RNA molecules includein vivo or in vitro RNA transcripts of the DNA molecules of the presentinvention. Isolated nucleic acid molecules according to the presentinvention further include such molecules produced synthetically.

[0074] Isolated nucleic acid molecules of the present invention includeDNA molecules comprising an open reading frame (ORF) with an initiationcodon at positions 89-91 of the nucleotide sequence shown in FIG. 1 (SEQID NO:1); DNA molecules comprising the coding sequence for the matureBAIT protein shown in FIG. 1 (amino acids 19-410) (SEQ ID NO:2); and DNAmolecules which comprise a sequence substantially different from thosedescribed above but which, due to the degeneracy of the genetic code,still encode the BAIT protein. Of course, the genetic code is well knownin the art. Thus, it would be routine for one skilled in the art togenerate the degenerate variants described above.

[0075] In another aspect, the invention provides isolated nucleic acidmolecules encoding the BAIT polypeptide having an amino acid sequenceencoded by the cDNA clone contained in the plasmid deposited as ATCCDeposit No. 97722. Preferably, this nucleic acid molecule will encodethe mature polypeptide encoded by the above-described deposited cDNAclone. The invention further provides an isolated nucleic acid moleculehaving the nucleotide sequence shown in FIG. 1 (SEQ ID NO:1) or thenucleotide sequence of the BAIT cDNA contained in the above-describeddeposited clone, or a nucleic acid molecule having a sequencecomplementary to one of the above sequences. Such isolated molecules,particularly DNA molecules, are useful as probes for gene mapping, by insitu hybridization with chromosomes, and for detecting expression of theBAIT gene in human tissue, for instance, by Northern blot analysis.

[0076] The present invention is further directed to nucleic acidmolecules encoding portions of the nucleotide sequences described hereinas well as to fragments of the isolated nucleic acid molecules describedherein. In particular, the invention provides a polynucleotide having anucleotide sequence representing the portion of SEQ ID NO:1 whichconsists of positions 1-410 of SEQ ID NO:1. In addition, the inventionprovides nucleic acid molecules having related nucleotide sequencesdetermined from the following related cDNA clones: HPBCT06R (SEQ IDNO:7), HBPDG64R (SEQ ID NO:8), and HPBCR79R (SEQ ID NO:9); see FIG. 4.More generally, by a fragment of an isolated nucleic acid moleculehaving the nucleotide sequence of the deposited cDNA or the nucleotidesequence shown in FIG. 1 (SEQ ED NO:1) is intended fragments at leastabout 15 nt, and more preferably at least about 20 nt, still morepreferably at least about 30 nt, and even more preferably, at leastabout 40 nt in length which are useful as diagnostic probes and primersas discussed herein. Of course, larger fragments 50-300 nt in length arealso useful according to the present invention as are fragmentscorresponding to most, if not all, of the nucleotide sequence of thedeposited cDNA or as shown in FIG. 1 (SEQ ID NO:1). By a fragment atleast 20 nt in length, for example, is intended fragments which include20 or more contiguous bases from the nucleotide sequence of thedeposited cDNA or the nucleotide sequence as shown in FIG. 1 (SEQ IDNO:1). Since the gene has been deposited and the nucleotide sequenceshown in FIG. 1 (SEQ ID NO:1) is provided, generating such DNA fragmentswould be routine to the skilled artisan. For example, restrictionendonuclease cleavage or shearing by sonication could easily be used togenerate fragments of various sizes. Alternatively, such fragments couldbe generated synthetically.

[0077] Preferred nucleic acid fragments of the present invention includenucleic acid molecules encoding epitope-bearing portions of the BAITpolypeptide as identified in FIG. 3 and described in more detail below.

[0078] In another aspect, the invention provides an isolated nucleicacid molecule comprising a polynucleotide which hybridizes understringent hybridization conditions, to a portion of the polynucleotidein a nucleic acid molecule of the invention described above, forinstance, the cDNA clone contained in ATCC Deposit 97722. By “stringenthybridization conditions” is intended overnight incubation at 42 C in asolution comprising: 50% formamide, 5× SSC (150 mM NaCl, 15 mM trisodiumcitrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10%dextran sulfate, and 20 g/ml denatured, sheared salmon sperm DNA,followed by washing the filters in 0.1× SSC at about 65 C.

[0079] By a polynucleotide which hybridizes to a “portion” of apolynucleotide is intended a polynucleotide (either DNA or RNA)hybridizing to at least about 15 nucleotides (nt), and more preferablyat least about 20 nt, still more preferably at least about 30 nt, andeven more preferably about 50-70 nt of the reference polynucleotide.These are useful as diagnostic probes and primers as discussed above andin more detail below.

[0080] Of course, polynucleotides hybridizing to a larger portion of thereference polynucleotide (e.g., the deposited cDNA clone), for instance,a portion 50-300 nt in length, or even to the entire length of thereference polynucleotide, are also useful as probes according to thepresent invention, as are polynucleotides corresponding to most, if notall, of the nucleotide sequence of the deposited cDNA or the nucleotidesequence as shown in FIG. 1 (SEQ ID NO:1). By a portion of apolynucleotides “at least 20 nt in length,” for example, is intended 20or more contiguous nucleotides from the nucleotide sequence of thereference polynucleotide (e.g., the deposited cDNA or the nucleotidesequence as shown in FIG. 1 (SEQ ID NO:1)). As indicated, such portionsare useful diagnostically either as a probe according to conventionalDNA hybridization techniques or as primers for amplification of a targetsequence by the polymerase chain reaction (PCR), as described, forinstance, in Molecular Cloning, A Laboratory Manual, 2nd. edition,Sambrook, J., Fritsch, E. F. and Maniatis, T., eds., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989), the entire disclosureof which is hereby incorporated herein by reference.

[0081] Since a BAIT cDNA clone has been deposited and its determinednucleotide sequence is provided in FIG. 1 (SEQ ID NO:1), generatingpolynucleotides which hybridize to a portion of the BAIT cDNA moleculewould be routine to the skilled artisan. For example, restrictionendonuclease cleavage or shearing by sonication of the BAIT cDNA clonecould easily be used to generate DNA portions of various sizes which arepolynucleotides that hybridize to a portion of the BAIT cDNA molecule.Alternatively, the hybridizing polynucleotides of the present inventioncould be generated synthetically according to known techniques. Ofcourse, a polynucleotide which hybridizes only to a poly A sequence(such as the 3 terminal poly(A) tract of the BAIT cDNA shown in FIG. 1(SEQ ID NO:1)), or to a complementary stretch of T (or U) residues,would not be included in a polynucleotide of the invention used tohybridize to a portion of a nucleic acid of the invention, since such apolynucleotide would hybridize to any nucleic acid molecule containing apoly (A) stretch or the complement thereof (e.g., practically anydouble-stranded cDNA clone).

[0082] As indicated, nucleic acid molecules of the present inventionwhich encode a BAIT polypeptide may include, but are not limited tothose encoding the amino acid sequence of the mature polypeptide, byitself; the coding sequence for the mature polypeptide and additionalsequences, such as those encoding the about 18 amino acid leader orsecretory sequence, such as a pre-, or pro- or prepro-protein sequence;the coding sequence of the mature polypeptide, with or without theaforementioned additional coding sequences, together with additional,non-coding sequences, including for example, but not limited to intronsand non-coding 5′ and 3′ sequences, such as the transcribed,non-translated sequences that play a role in transcription, mRNAprocessing, including splicing and polyadenylation signals, forexample—ribosome binding and stability of mRNA; an additional codingsequence which codes for additional amino acids, such as those whichprovide additional functionalities.

[0083] Thus, the sequence encoding the polypeptide may be fused to amarker sequence, such as a sequence encoding a peptide which facilitatespurification of the fused polypeptide. In certain preferred embodimentsof this aspect of the invention, the marker amino acid sequence is ahexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc.), among others, many of which are commercially available.As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824(1989), for instance, hexa-histidine provides for convenientpurification of the fusion protein. The “HA” tag is another peptideuseful for purification which corresponds to an epitope derived from theinfluenza hemagglutinin protein, which has been described by Wilson etal., Cell 37:767 (1984). As discussed below, other such fusion proteinsinclude the BAIT fused to Fc at the N- or C-terminus.

[0084] The present invention further relates to variants of the nucleicacid molecules of the present invention, which encode portions, analogsor derivatives of the BAIT protein. Variants may occur naturally, suchas a natural allelic variant. By an “allelic variant” is intended one ofseveral alternate forms of a gene occupying a given locus on achromosome of an organism. GenesIII, Lewin, B., ed., John Wiley & Sons,New York (1985). Non-naturally occurring variants may be produced usingart-known mutagenesis techniques.

[0085] Such variants include those produced by nucleotide substitutions,deletions or additions. The substitutions, deletions or additions mayinvolve one or more nucleotides. The variants may be altered in codingregions, non-coding regions, or both. Alterations in the coding regionsmay produce conservative or non-conservative amino acid substitutions,deletions or additions. Especially preferred among these are silentsubstitutions, additions and deletions, which do not alter theproperties and activities of the BAIT protein or portions thereof. Alsoespecially preferred in this regard are conservative substitutions. Mosthighly preferred are nucleic acid molecules encoding the mature proteinhaving the amino acid sequence shown in FIG. 1 (SEQ ID NO:2) or themature BAIT amino acid sequence encoded by the deposited cDNA clone.

[0086] Further embodiments of the invention include isolated nucleicacid molecules comprising a polynucleotide having a nucleotide sequenceat least 99% identical, and more preferably at least 99.1% 99.9%identical to (a) a nucleotide sequence encoding the full-length BAITpolypeptide having the complete amino acid sequence in FIG. 1 (SEQ IDNO:2), including the leader sequence; (b) a nucleotide sequence encodingthe mature BAIT polypeptide (full-length polypeptide with the leaderremoved) having the amino acid sequence at positions 19-94 in FIG. 1(SEQ ID NO:2); (c) a nucleotide sequence encoding the full-length BAITpolypeptide having the complete amino acid sequence including the leaderencoded by the cDNA clone contained in ATCC Deposit No. 97722; (d) anucleotide sequence encoding the mature BAIT polypeptide having theamino acid sequence encoded by the cDNA clone contained in ATCC DepositNo. 97722; or (e) a nucleotide sequence complementary to any of thenucleotide sequences in (a), (b), (c) or (d).

[0087] By a polynucleotide having a nucleotide sequence at least, forexample, 99% “identical” to a reference nucleotide sequence encoding aBAIT polypeptide is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to one point mutations per each100 nucleotides of the reference nucleotide sequence encoding the BAITpolypeptide. In other words, to obtain a polynucleotide having anucleotide sequence at least 99% identical to a reference nucleotidesequence, up to 1% of the nucleotides in the reference sequence may bedeleted or substituted with another nucleotide, or a number ofnucleotides up to 1% of the total nucleotides in the reference sequencemay be inserted into the reference sequence. These mutations of thereference sequence may occur at the 5 or 3 terminal positions of thereference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence.

[0088] As a practical matter, whether any particular nucleic acidmolecule is at least 99.1%, to 99.9% identical to, for instance, thenucleotide sequence shown in FIG. 1 or to the nucleotides sequence ofthe deposited cDNA clone can be determined conventionally using knowncomputer programs such as the Bestfit program (Wisconsin SequenceAnalysis Package, Version 8 for Unix, Genetics Computer Group,University Research Park, 575 Science Drive, Madison, Wis. 53711).Bestfit uses the local homology algorithm of Smith and Watennan,Advances in Applied Mathematics 2:482-489 (1981), to find the bestsegment of homology between two sequences. When using Bestfit or anyother sequence alignment program to determine whether a particularsequence is, for instance, 99% identical to a reference sequenceaccording to the present invention, the parameters are set, of course,such that the percentage of identity is calculated over the full lengthof the reference nucleotide sequence and that gaps in homology of up to1% of the total number of nucleotides in the reference sequence areallowed.

[0089] The present application is directed to nucleic acid molecules atleast 96%, 96.1%, 96.2%, 96.3% to 99.9% identical to the nucleic acidsequence shown in FIG. 1 (SEQ ID NO:1) or to the nucleic acid sequenceof the deposited cDNA, irrespective of whether they encode a polypeptidehaving BAIT activity. This is because even where a particular nucleicacid molecule does not encode a polypeptide having BAIT activity, one ofskill in the art would still know how to use the nucleic acid molecule,for instance, as a hybridization probe or a polymerase chain reaction(PCR) primer. Uses of the nucleic acid molecules of the presentinvention that do not encode a polypeptide having BAIT activity include,inter alia, (1) isolating the BAIT gene or allelic variants thereof in acDNA library; (2) in situ hybridization (e.g., “FISH”) to metaphasechromosomal spreads to provide precise chromosomal location of the BAITgene, as described in Verma et al., Human Chromosomes: A Manual of BasicTechniques, Pergamon Press, New York (1988); and Northern Blot analysisfor detecting BAIT mRNA expression in specific tissues.

[0090] Preferred, however, are nucleic acid molecules having sequencesat least 99%, to 99.9% identical to the nucleic acid sequence shown inFIG. 1 (SEQ ID NO:1) or to the nucleic acid sequence of the depositedcDNA which do, in fact, encode a polypeptide having BAIT proteinactivity. By “a polypeptide having BAIT activity” is intendedpolypeptides exhibiting activity similar, but not necessarily identical,to an activity of the BAIT protein of the invention (either thefull-length protein or, preferably, the mature protein), as measured ina particular biological assay. For example, the BAIT protein of thepresent invention inhibits the proteolytic activity of tissue-typeplasminogen activator (t-PA). Briefly, the assay involves measuring theinhibitory activity against various proteases, particularly tPA, using asingle step chromogenic assay essentially as described (Lawrence, D. A.,et. al. (1990) The Journal of Biological Chemistry, 265, 20293-20301).

[0091] BAIT protein inhibits proteolytic activity of t-PA in adose-dependent manner in the above-described assay. Thus, “a polypeptidehaving BAIT protein activity” includes polypeptides that also exhibitany of the same t-PA-inhibiting activities in the above-described assayin a dose-dependent manner. Although the degree of dose dependentactivity need not be identical to that of the BAIT protein, preferably,“a polypeptide having BAIT protein activity” will exhibit substantiallysimilar dosedependence in a given activity as compared to the BAITprotein (i.e., the candidate polypeptide will exhibit greater activityor not more than about 25-fold less and, preferably, not more than abouttenfold less activity relative to the reference BAIT protein).

[0092] Of course, due to the degeneracy of the genetic code, one ofordinary skill in the art will immediately recognize that a large numberof the nucleic acid molecules having a sequence at least 99% identicalto the nucleic acid sequence of the deposited cDNA or the nucleic acidsequence shown in FIG. 1 (SEQ ID NO:1) will encode a polypeptide “havingBAIT protein activity.” In fact, since degenerate variants of thesenucleotide sequences all encode the same polypeptide, this will be clearto the skilled artisan even without performing the above describedcomparison assay. It will be further recognized in the art that, forsuch nucleic acid molecules that are not degenerate variants, areasonable number will also encode a polypeptide having BAIT proteinactivity. This is because the skilled artisan is fully aware of aminoacid substitutions that are either less likely or not likely tosignificantly effect protein function (e.g., replacing one aliphaticamino acid with a second aliphatic amino acid), as further describedbelow.

[0093] BAIT Polypeptides and Fragments

[0094] The invention further provides an isolated BAIT polypeptidehaving the amino acid sequence encoded by the deposited cDNA, or theamino acid sequence in FIG. 1 (SEQ ID NO:2), or a peptide or polypeptidecomprising a portion of the above polypeptides. The terms “peptide” and“oligopeptide” are considered synonymous (as is commonly recognized)-andeach term can be used interchangeably as the context requires toindicate a chain of at least two amino acids coupled by peptidyllinkages. The word “polypeptide” is used herein for chains containingmore than ten amino acid residues. All oligopeptide and polypeptideformulas or sequences herein are written from left to right and in thedirection from amino terminus to carboxy terminus.

[0095] In addition to mature and N-terminal deletion forms of theprotein discussed above, it will be recognized by one of ordinary skillin the art that some amino acid sequences of the BAIT polypeptide can bevaried without significant effect of the structure or function of theprotein. If such differences in sequence are contemplated, it should beremembered that there will be critical areas on the protein whichdetermine activity. In general, it is possible to replace residues whichform the tertiary structure, provided that residues performing a similarfunction are used. In other instances, the type of residue may becompletely unimportant if the alteration occurs at a non-critical regionof the protein.

[0096] Thus, the invention further includes variations of the BAITpolypeptide which show substantial BAIT polypeptide activity or whichinclude regions of BAIT protein such as the protein portions discussedbelow. Such mutants include deletions, insertions, inversions, repeats,and type substitutions selected according to general rules known in theart so as have little effect on activity.

[0097] For example, guidance concerning how to make phenotypicallysilent amino acid substitutions is provided in Bowie et al.,“Deciphering the Message in Protein Sequences: Tolerance to Amino AcidSubstitutions,” Science 247:1306-1310 (1990), wherein the authorsindicate that there are two main strategies for studying the toleranceof an amino acid sequence to change.

[0098] The first strategy exploits the tolerance of amino acidsubstitutions by natural selection during the process of evolution. Bycomparing amino acid sequences in different species, conserved aminoacids can be identified. These conserved amino acids are likelyimportant for protein function. In contrast, the amino acid positionswhere substitutions have been tolerated by natural selection indicatesthat these positions are not critical for protein function. Thus,positions tolerating amino acid substitution could be modified whilestill maintaining biological activity of the protein.

[0099] The second strategy uses genetic engineering to introduce aminoacid changes at specific positions of a cloned gene to identify regionscritical for protein function. For example, site directed mutagenesis oralanine-scanning mutagenesis (introduction of single alanine mutationsat every residue in the molecule) can be used. (Cunningham and Wells,Science 244:1081-1085 (1989).) The resulting mutant molecules can thenbe tested for biological activity.

[0100] As the authors state, these two strategies have revealed thatproteins are surprisingly tolerant of amino acid substitutions. Theauthors further indicate which amino acid changes are likely to bepermissive at certain amino acid positions in the protein. For example,most buried (within the tertiary structure of the protein) amino acidresidues require nonpolar side chains, whereas few features of surfaceside chains are generally conserved. Moreover, tolerated conservativeamino acid substitutions involve replacement of the aliphatic orhydrophobic amino acids Ala, Val, Leu and Ile; replacement of thehydroxyl residues Ser and Thr; replacement of the acidic residues Aspand Glu; replacement of the amide residues Asn and Gln, replacement ofthe basic residues Lys, Arg, and His; replacement of the aromaticresidues Phe, Tyr, and Trp, and replacement of the small-sized aminoacids Ala, Ser, Thr, Met, and Gly.

[0101] Of course, due to the degeneracy of the genetic code, one ofordinary skill in the art will immediately recognize that a large numberof the nucleic acid molecules having a sequence at least 90%, 95%, 96%,97%, 98%, or 99% identical to the nucleic acid sequence of the depositedcDNA, the nucleic acid sequence shown in FIG. 1 (SEQ ID NO:1), orfragments thereof, will encode polypeptides “having BAIT functionalactivity.” In fact, since degenerate variants of any of these nucleotidesequences all encode the same polypeptide, in many instances, this willbe clear to the skilled artisan even without performing the abovedescribed comparison assay. It will be further recognized in the artthat, for such nucleic acid molecules that are not degenerate variants,a reasonable number will also encode a polypeptide having BAITfunctional activity. This is because the skilled artisan is fully awareof amino acid substitutions that are either less likely or not likely tosignificantly effect protein function (e.g., replacing one aliphaticamino acid with a second aliphatic amino acid), as further describedbelow.

[0102] For example, site directed changes at the amino acid level ofBAIT can be made by replacing a particular amino acid with aconservative amino acid. Preferred conservative mutations include: M1replaced with A, G, I, L, S, T, or V; A2 replaced with G, I, L, S, T, M,or V; F3 replaced with W, or Y; L4 replaced with A, G, I, S, T, M, or V;G5 replaced with A, I, L, S, T, M, or V; L6 replaced with A, G, I, S, T,M, or V; F7 replaced with W, or Y; S8 replaced with A, G, I, L, T, M, orV; L9 replaced with A, G, I, S, T, M, or V; L10 replaced with A, G, I,S, T, M, or V; V11 replaced with A, G, I, L, S, T, or M; L12 replacedwith A, G, I, S, T, M, or V; Q13 replaced with N; S14 replaced with A,G, I, L, T, M, or V; M15 replaced with A, G, I, L, S, T, or V; A16replaced with G, I, L, S, T, M, or V; T17 replaced with A, G, I, L, S,M, or V; G18 replaced with A, I, L, S, T, M, or V; A19 replaced with G,I, L, S, T, M, or V; T20 replaced with A, G, I, L, S, M, or V; F21replaced with W, or Y; E23 replaced with D; E24 replaced with D; A25replaced with G, I, L, S, T, M, or V; 126 replaced with A, G, L, S, T,M, or V; A27 replaced with G, I, L, S, T, M, or V; D28 replaced with E;L29 replaced with A, G, I, S, T, M, or V; S30 replaced with A, G, I, L,T, M, or V; V31 replaced with A, G, I, L, S, T, or M; N32 replaced withQ; M33 replaced with A, G, I, L, S, T, or V; Y34 replaced with F, or W;N35 replaced with Q; R36 replaced with H, or K; L37 replaced with A, G,I, S, T, M, or V; R38 replaced with H, or K; A39 replaced with G, I, L,S, T, M, or V; T40 replaced with A, G, I, L, S, M, or V; G41 replacedwith A, I, L, S, T, M, or V; E42 replaced with D; D43 replaced with E;E44 replaced with D; N45 replaced with Q; 146 replaced with A, G, L, S,T, M, or V; L47 replaced with A, G, I, S, T, M, or V; F48 replaced withW, or Y; S49 replaced with A, G, I, L, T, M, or V; L51 replaced with A,G, I, S, T, M, or V; S52 replaced with A, G, I, L, T, M, or V; 153replaced with A, G, L, S, T, M, or V; A54 replaced with G, I, L, S, T,M, or V; L55 replaced with A, G, I, S, T, M, or V; A56 replaced with G,I, L, S, T, M, or V; M57 replaced with A, G, I, L, S, T, or V; G58replaced with A, I, L, S, T, M, or V; M59 replaced with A, G, I, L, S,T, or V; M60 replaced with A, G, I, L, S, T, or V; E61 replaced with D;L62 replaced with A, G, I, S, T, M, or V; G63 replaced with A, I, L, S,T, M, or V; A64 replaced with G,I, L, S, T, M, or V; Q65 replaced withN; G66 replaced with A, I, L, S, T, M, or V; S67 replaced with A, G, I,L, T, M, or V; T68 replaced with A, G, I, L, S, M, or V; Q69 replacedwith N; K70 replaced with H, or R; E71 replaced with D; I72 replacedwith A, G, L, S, T, M, or V; R73 replaced with H, or K; H74 replacedwith K, or R; S75 replaced with A, G, I, L, T, M, or V; M76 replacedwith A, G, I, L, S, T, or V; G77 replaced with A, I, L, S, T, M, or V;Y78 replaced with F, or W; D79 replaced with E; S80 replaced with A, G,I, L, T, M, or V; L81 replaced with A, G, I, S, T, M, or V; K82 replacedwith H, or R; N83 replaced with Q; G84 replaced with A, I, L, S, T, M,or V; E85 replaced with D; E86 replaced with D; F87 replaced with W, orY; S88 replaced with A, G, I, L, T, M, or V; F89 replaced with W, or Y;L90 replaced with A, G, I, S, T, M, or V; K91 replaced with H, or R; E92replaced with D; F93 replaced with W, or Y; S94 replaced with A, G, I,L, T, M, or V; N95 replaced with Q; M96 replaced with A, G, I, L, S, T,or V; V97 replaced with A, G, I, L, S, T, or M; T98 replaced with A, G,I, L, S, M, or V; A99 replaced with G, I, L, S, T, M, or V; K100replaced with H, or R; E101 replaced with D; S102 replaced with A, G, 1,L, T, M, or V; Q103 replaced with N; Y104 replaced with F, or W; V105replaced with A, G, I, L, S, T, or M; M106 replaced with A, G, I, L, S,T, or V; K107 replaced with H, or R; I108 replaced with A, G, L, S, T,M, or V; A109 replaced with G, I, L, S, T, M, or V; N110 replaced withQ; S111 replaced with A, G, I, L, T, M, or V; L112 replaced with A, G,I, S, T, M, or V; F113 replaced with W, or Y; V114 replaced with A, G,I, L, S, T, or M; Q115 replaced with N; N116 replaced with Q; G117replaced with A, I, L, S, T, M, or V; F118 replaced with W, or Y; H119replaced with K, or R; V120 replaced with A, G, I, L, S, T, or M; N121replaced with Q; E122 replaced with D; E123 replaced with D; F124replaced with W, or Y; L125 replaced with A, G, I, S, T, M, or V; Q126replaced with N; M127 replaced with A, G, I, L, S, T, or V; M128replaced with A, G, I, L, S, T, or V; K129 replaced with H, or R; K130replaced with H, or R; Y131 replaced with F, or W; F132 replaced with W,or Y; N133 replaced with Q; A134 replaced with G, I, L, S, T, M, or V;A135 replaced with G, I, L, S, T, M, or V; V136 replaced with A, G, I,L, S, T, or M; N137 replaced with Q; H138 25. replaced with K, or R;V139 replaced with A, G, I, L, S, T, or M; D140 replaced with E; F141replaced with W, or Y; S142 replaced with A, G, I, L, T, M, or V; Q143replaced with N; N144 replaced with Q; V145 replaced with A, G, I, L, S,T, or M; A146 replaced with G, I, L, S, T, M, or V; V147 replaced withA, G, I, L, S, T, or M; A148 replaced with G, I, L, S, T, M, or V; N149replaced with Q; Y150 replaced with F, or W; I151 replaced with A, G, L,S, T, M, or V; N152 replaced with Q; K153 replaced with H, or R; W154replaced with F, or Y; V155 replaced with A, G, I, L, S, T, or M; E156replaced with D; N157 replaced with Q; N158 replaced with Q; T159replaced with A, G, I, L, S, M, or V; N160 replaced with Q; N161replaced with Q; L162 replaced with A, G, I, S, T, M, or V; V163replaced with A, G, I, L, S, T, or M; K164 replaced with H, or R; D165replaced with E; L166 replaced with A, G, I, S, T, M, or V; V167replaced with A, G, I, L, S, T, or M; S168 replaced with A, G, I, L, T,M, or V; R170 replaced with H, or K; D171 replaced with E; F172 replacedwith W, or Y; D173 replaced with E; A174 replaced with G, I, L, S, T, M,or V; A175 replaced with G, I, L, S, T, M, or V; T176 replaced with A,G, I, L, S, M, or V; Y177 replaced with F, or W; L178 replaced with A,G, I, S, T, M, or V; A179 replaced with G, I, L, S, T, M, or V; L180replaced with A, G, I, S, T, M, or V; I181 replaced with A, G, L, S, T,M, or V; N182 replaced with Q; A183 replaced with G, I, L, S, T, M, orV; V184 replaced with A, G, I, L, S, T, or M; Y185 replaced with F, orW; F186 replaced with W, or Y; K187 replaced with H, or R; G188 replacedwith A, I, L, S, T, M, or V; N189 replaced with Q; W190 replaced with F,or Y; K191 replaced with H, or R; S192 replaced with A, G, I, L, T, M,or V; Q193 replaced with N; F194 replaced with W, or Y; R195 replacedwith H, or K; E197 replaced with D; N198 replaced with Q; T199 replacedwith A, G, I, L, S, M, or V; R200 replaced with H, or K; T201 replacedwith A, G, I, L, S, M, or V; F202 replaced with W, or Y; S203 replacedwith A, G, I, L, T, M, or V; F204 replaced with W, or Y; T205 replacedwith A, G, I, L, S, M, or V; K206 replaced with H, or R; D207 replacedwith E; D208 replaced with E; E209 replaced with D; S210 replaced withA, G, I, L, T, M, or V; E211 replaced with D; V212 replaced with A, G,I, L, S, T, or M; Q213 replaced with N; I214 replaced with A, G, L, S,T, M, or V; M216 replaced with A, G, I, L, S, T, or V; M217 replacedwith A, G, I, L, S, T, or V; Y218 replaced with F, or W; Q219 replacedwith N; Q220 replaced with N; G221 replaced with A, I, L, S, T, M, or V;E222 replaced with D; F223 replaced with W, or Y; Y224 replaced with F,or W; Y225 replaced with F, or W; G226 replaced with A, I, L, S, T, M,or V; E227 replaced with D; F228 replaced with W, or Y; S229 replacedwith A, G, I, L, T, M, or V; D230 replaced with E; G231 replaced with A,I, L, S, T, M, or V; S232 replaced with A, G, I, L, T, M, or V; N233replaced with Q; E234 replaced with D; A235 replaced with G, I, L, S, T,M, or V; G236 replaced with A, I, L, S, T, M, or V; G237 replaced withA, I, L, S, T, M, or V; I238 replaced with A, G, L, S, T, M, or V; Y239replaced with F, or W; Q240 replaced with N; V241 replaced with A, G, I,L, S, T, or M; L242 replaced with A, G, I, S, T, M, or V; E243 replacedwith D; I244 replaced with A, G, L, S, T, M, or V; Y246 replaced with F,or W; E247 replaced with D; G248 replaced with A, I, L, S, T, M, or V;D249 replaced with E; E250 replaced with D; I251 replaced with A, G, L,S, T, M, or V; S252 replaced with A, G, I, L, T, M, or V; M253 replacedwith A, G, I, L, S, T, or V; M254 replaced with A, G, I, L, S, T, or V;L255 replaced with A, G, I, S, T, M, or V; V256 replaced with A, G, I,L, S, T, or M; L257 replaced with A, G, I, S, T, M, or V; S258 replacedwith A, G, I, L, T, M, or V; R259 replaced with H, or K; Q260 replacedwith N; E261 replaced with D; V262 replaced with A, G, I, L, S, T, or M;L264 replaced with A, G, I, S, T, M, or V; A265 replaced with G, I, L,S, T, M, or V; T266 replaced with A, G, I, L, S, M, or V; L267 replacedwith A, G, I, S, T, M, or V; E268 replaced with D; L270 replaced with A,G, I, S, T, M, or V; V271 replaced with A, G, I, L, S, T, or M; K272replaced with H, or R; A273 replaced with G, I, L, S, T, M, or V; Q274replaced with N; L275 replaced with A, G, I, S, T, M, or V; V276replaced with A, G, I, L, S, T, or M; E277 replaced with D; E278replaced with D; W279 replaced with F, or Y; A280 replaced with G, I, L,S, T, M, or V; N281 replaced with Q; S282 replaced with A, G, I, L, T,M, or V; V283 replaced with A, G, I, L, S, T, or M; K284 replaced withH, or R; K285 replaced with H, or R; Q286 replaced with N; K287 replacedwith H, or R; V288 replaced with A, G, I, L, S, T, or M; E289 replacedwith D; V290 replaced with A, G, I, L, S, T, or M; Y291 replaced with F,or W; L292 replaced with A, G, I, S, T, M, or V; R294 replaced with H,or K; F295 replaced with W, or Y; T296 replaced with A, G, I, L, S, M,or V; V297 replaced with A, G, I, L, S, T, or M; E298 replaced with D;Q299 replaced with N; E300 replaced with D; I301 replaced with A, G, L,S, T, M, or V; D302 replaced with E; L303 replaced with A, G, I, S, T,M, or V; K304 replaced with H, or R; D305 replaced with E; V306 replacedwith A, G, I, L, S, T, or M; L307 replaced with A, G, I, S, T, M, or V;K308 replaced with H, or R; A309 replaced with G, I, L, S, T, M, or V;L310 replaced with A, G, I, S, T, M, or V; G311 replaced with A, I, L,S, T, M, or V; I312 replaced with A, G, L, S, T, M, or V; T313 replacedwith A, G, I, L, S, M, or V; E314 replaced with D; I315 replaced with A,G, L, S, T, M, or V; F316 replaced with W, or Y; I317 replaced with A, G, L, S, T, M, or V; K318 replaced with H, or R; D 319 replaced with E;A320 replaced with G, I, L, S, T, M, or V; N321 replaced with Q; L322replaced with A, G, L, S, T, M, or V; T323 replaced with A, G, I, L, S,M, or V; G324 replaced with A, I, L, S, T, M, or V; L325 replaced withA, G, I, S, T, M, or V; S326 replaced with A, G, I, L, T, M, or V; D327replaced with E; N328 replaced with Q; K329 replaced with H, or R; E330replaced with D; I331 replaced with A, G, L, S, T, M, or V; F332replaced with W, or Y; L333 replaced with A, G, I, S, T, M, or V; S334replaced with A, G, I, L, T, M, or V; K335 replaced with H, or R; A336replaced with G, I, L, S, T, M, or V; I337 replaced with A, G, L, S, T,M, or V; H338 replaced with K, or R; K339 replaced with H, or R; S340replaced with A, G, I, L, T, M, or V; F341 replaced with W, or Y; L342replaced with A, G, I, S, T, M, or V; E343 replaced with D; V344replaced with A, G, I, L, S, T, or M; N345 replaced with Q; E346replaced with D; E347 replaced with D; G348 replaced with A, I, L, S, T,M, or V; S349 replaced with A, G, I, L, T, M, or V; E350 replaced withD; A351 replaced with G, I, L, S, T, M, or V; A352 replaced with G, I,L, S, T, M, or V; A353 replaced with G, I, L, S, T, M, or V; V354replaced with A, G, I, L, S, T, or M; S355 replaced with A, G, I, L, T,M, or V; G356 replaced with A, I, L, S, T, M, or V; M357 replaced withA, G, I, L, S, T, or V; I358 replaced with A, G, L, S, T, M, or V; A359replaced with G, I, L, S, T, M, or V; I360 replaced with A, G, L, S, T,M, or V; S361 replaced with A, G, I, L, T, M, or V; R362 replaced withH, or K; M363 replaced with A, G, I, L, S, T, or V; A364 replaced withG, I, L, S, T, M, or V; V365 replaced with A, G, I, L, S, T, or M; L366replaced with A, G, I, S, T, M, or V; Y367 replaced with F, or W; Q369replaced with N; V370 replaced with A, G, I, L, S, T, or M; I371replaced with A, G, L, S, T, M, or V; V372 replaced with A, G, I, L, S,T, or M; D373 replaced with E; H374 replaced with K, or R; F376 replacedwith W, or Y; F377 replaced with W, or Y; F378 replaced with W, or Y;L379 replaced with A, G, I, S, T, M, or V; I380 replaced with A, G, L,S, T, M, or V; R381 replaced with H, or K; N382 replaced with Q; R383replaced with H, or K; R384 replaced with H, or K; T385 replaced with A,G, I, L, S, M, or V; G386 replaced with A, I, L, S, T, M, or V; T387replaced with A, G, I, L, S, M, or V; I388 replaced with A, G, L, S, T,M, or V; L389 replaced with A, G, I, S, T, M, or V; F390 replaced withW, or Y; M391 replaced with A, G, I, L, S, T, or V; G392 replaced withA, I, L, S, T, M, or V; R393 replaced with H, or K; V394 replaced withA, G, I, L, S, T, or M; M395 replaced with A, G, I, L, S, T, or V; H396replaced with K, or R; E398 replaced with D; T399 replaced with A, G, I,L, S, M, or V; M400 replaced with A, G, I, L, S, T, or V; N401 replacedwith Q; T402 replaced with A, G, I, L, S, M, or V; S403 replaced with A,G, I, L, T, M, or V; G404 replaced with A, I, L, S, T, M, or V; H405replaced with K, or R; D406 replaced with E; F407 replaced with W, or Y;E408 replaced with D; E409 replaced with D; and/or L410 replaced with A,G, I, S, T, M, or V of SEQ ID NO:2.

[0103] The resulting constructs can be routinely screened for activitiesor functions described throughout the specification and known in theart. Preferably, the resulting constructs have an increased and/or adecreased BAIT activity or function, while the remaining BAIT activitiesor functions are maintained. More preferably, the resulting constructshave more than one increased and/or decreased BAIT activity or function,while the remaining BAIT activities or functions are maintained.

[0104] Besides conservative amino acid substitution, variants of BAITinclude (i) substitutions with one or more of the non-conserved aminoacid residues, where the substituted amino acid residues may or may notbe one encoded by the genetic code, or (ii) substitution with one ormore of amino acid residues having a substituent group, or (iii) fusionof the mature polypeptide with another compound, such as a compound toincrease the stability and/or solubility of the polypeptide (forexample, polyethylene glycol), or (iv) fusion of the polypeptide withadditional amino acids, such as, for example, an IgG Fc fusion regionpeptide, or leader or secretory sequence, or a sequence facilitatingpurification. Such variant polypeptides are deemed to be within thescope of those skilled in the art from the teachings herein.

[0105] For example, BAIT polypeptide variants containing amino acidsubstitutions of charged amino acids with other charged or neutral aminoacids may produce proteins with improved characteristics, such as lessaggregation. Aggregation of pharmaceutical formulations both reducesactivity and increases clearance due to the aggregate's immunogenicactivity. (Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967);Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev.Therapeutic Drug Carrier Systems 10:307-377 (1993).)

[0106] For example, preferred non-conservative substitutions of BAITinclude: M1 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A2replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F3 replaced with D,E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; L4 replaced with D,E, H, K, R, N, Q, F, W, Y, P, or C; G5 replaced with D, E, H, K, R, N,Q, F, W, Y, P, or C; L6 replaced with D, E, H, K, R, N, Q, F, W, Y, P,or C; F7 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P,or C; S8 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L9replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L10 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; V11 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; L12 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; Q13 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W,Y, P, or C; S14 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; M15replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A16 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; T17 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; G18 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; A19 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T20replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F21 replaced withD, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; P22 replaced withD, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; E23 replacedwith H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E24replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;A25 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; 126 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; A27 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; D28 replaced with H, K, R, A, G, I, L, S,T, M, V, N, Q, F, W, Y, P, or C; L29 replaced with D, E, H, K, R, N, Q,F, W, Y, P, or C; S30 replaced with D, E, H, K, R, N, Q, F, W, Y, P, orC; V31 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N32 replacedwith D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; M33replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y34 replaced withD, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; N35 replaced withD, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; R36 replacedwith D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L37 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; R38 replaced with D, E, A,G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A39 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; T40 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; G41 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;E42 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; D43 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,or C; E44 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,P, or C; N45 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W,Y, P, or C; I46 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L47replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F48 replaced withD, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; S49 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; P50 replaced with D, E, H, K, R,A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; L51 replaced with-D, E, H,K, R, N, Q, F, W, Y, P, or C; S52 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; 153 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;A54 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L55 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; A56 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; M57 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; G58 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;M59 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; M60 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; E61 replaced with H, K, R,A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L62 replaced with D, E,H, K, R, N, Q, F, W, Y, P, or C; G63 replaced with D, E, H, K, R, N, Q,F, W, Y, P, or C; A64 replaced with D, E, H, K, R, N, Q, F, W, Y, P, orC; Q65 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P,or C; G66 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S67replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T68 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; Q69 replaced with D, E, H, K, R,A, G, I, L, S, T, M, V, F, W, Y, P, or C; K70 replaced with D, E, A, G,I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E71 replaced with H, K, R, A,G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; I72 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; R73 replaced with D, E, A, G, I, L, S, T,M, V, N, Q, F, W, Y, P, or C; H74 replaced with D, E, A, G, I, L, S, T,M, V, N, Q, F, W, Y, P, or C; S75 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; M76 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;G77 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y78 replacedwith D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; D79 replacedwith H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S80replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L81 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; K82 replaced with D, E, A, G, I,L, S, T, M, V, N, Q, F, W, Y, P, or C; N83 replaced with D, E, H, K, R,A, G, I, L, S, T, M, V, F, W, Y, P, or C; G84 replaced with D, E, H, K,R, N, Q, F, W, Y, P, or C; E85 replaced with H, K, R, A, G, I, L, S, T,M, V, N, Q, F, W, Y, P, or C; E86 replaced with H, K, R, A, G, I, L, S,T, M, V, N, Q, F, W, Y, P, or C; F87 replaced with D, E, H, K, R, N, Q,A, G, I, L, S, T, M, V, P, or C; S88 replaced with D, E, H, K, R, N, Q,F, W, Y, P, or C; F89 replaced with D, E, H, K, R, N, Q, A, G, I, L, S,T, M, V, P, or C; L90 replaced with D, E, H, K, R, N, Q, F, W, Y, P, orC; K91 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; E92 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,or C; F93 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P,or C; S94 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N95replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C;M96 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V97 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; T98 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; A99 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; K100 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,W, Y, P, or C; E101 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q,F, W, Y, P, or C; S102 replaced with D, E, H, K, R, N, Q, F, W, Y, P, orC; Q103 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P,or C; Y104 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P,or C; V105 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; M106replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K107 replaced withD, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; I108 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; A109 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; N110 replaced with D, E, H, K, R, A, G, I, L, S,T, M, V, F, W, Y, P, or C; S111 replaced with D, E, H, K, R, N, Q, F, W,Y, P, or C; L112 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;F113 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;VI 14 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q115 replacedwith D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; N116replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; G17 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F118 replacedwith D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; Hl 19replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V120replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N121 replaced withD, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; E122 replacedwith H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E123replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;F124 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;L125 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q126 replacedwith D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; M127replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; M128 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; K129 replaced with D, E, A, G, I,L, S, T, M, V, N, Q, F, W, Y, P, or C; K130 replaced with D, E, A, G, I,L, S, T, M, V, N, Q, F, W, Y, P, or C; Y131 replaced with D, E, H, K, R,N, Q, A, G, I, L, S, T, M, V, P, or C; F132 replaced with D, E, H, K, R,N, Q, A, G, I, L, S, T, M, V, P, or C; N133 replaced with D, E, H, K, R,A, G, I, L, S, T, M, V, F, W, Y, P, or C; A134 replaced with D, E, H, K,R, N, Q, F, W, Y, P, or C; A135 replaced with D, E, H, K, R, N, Q, F, W,Y, P, or C; V136 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;N137 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, orC; H138 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; V139 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D140replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;F141 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;S142 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q143 replacedwith D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; N144replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C;V145 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A146 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; V147 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; A148 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; N149 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,F, W, Y, P, or C; Y150 replaced with D, E, H, K, R, N, Q, A, G, I, L, S,T, M, V, P, or C; I151 replaced with D, E, H, K, R, N, Q, F, W, Y, P, orC; N152 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P,or C; K153 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,or C; W154 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P,or C; V155 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E156replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;N157 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, orC; N158 replaced with D, E, B, K, R, A, G, I, L, S, T, M, V, F, W, Y, P,or C; T159 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N160replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C;N161 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, orC; L162 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V163replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K164 replaced withD, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; D165 replaced withH, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L166 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; V167 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; S168 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; P169 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,N, Q, F, W, Y, or C; R170 replaced with D, E, A, G, I, L, S, T, M, V, N,Q, F, W, Y, P, or C; D171 replaced with H, K, R, A, G, I, L, S, T, M, V,N, Q, F, W, Y, P, or C; F172 replaced with D, E, H, K, R, N, Q, A, G, I,L, S, T, M, V, P, or C; D173 replaced with H, K, R, A, G, I, L, S, T, M,V, N, Q, F, W, Y, P, or C; A174 replaced with D, E, H, K, R, N, Q, F, W,Y, P, or C; A175 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;T176 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y177 replacedwith D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; L178 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; A179 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; L180 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; I181 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;N182 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, orC; A183 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V184replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y185 replaced withD, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; F186 replaced withD, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; K187 replaced withD, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G188 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; N189 replaced with D, E, H, K, R,A, G, I, L, S, T, M, V, F, W, Y, P, or C; W190 replaced with D, E, H, K,R, N, Q, A, G, I, L, S, T, M, V, P, or C; K191 replaced with D, E, A, G,I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S192 replaced with D, E, H, K,R, N, Q, F, W, Y, P, or C; Q193 replaced with D, E, H, K, R, A, G, I, L,S, T, M, V, F, W, Y, P, or C; F194 replaced with D, E, H, K, R, N, Q, A,G, I, L, S, T, M, V, P, or C; R195 replaced with D, E, A, G, I, L, S, T,M, V, N, Q, F, W, Y, P, or C; P196 replaced with D, E, H, K, R, A, G, I,L, S, T, M, V, N, Q, F, W, Y, or C; E197 replaced with H, K, R, A, G, I,L, S, T, M, V, N, Q, F, W, Y, P, or C; N198 replaced with D, E, H, K, R,A, G, I, L, S, T, M, V, F, W, Y, P, or C; T199 replaced with D, E, H, K,R, N, Q, F, W, Y, P, or C; R200 replaced with D, E, A, G, I, L, S, T, M,V, N, Q, F, W, Y, P, or C; T201 replaced with D, E, H, K, R, N, Q, F, W,Y, P, or C; F202 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M,V, P, or C; S203 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;F204 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;T205 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K206 replacedwith D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; D207 replacedwith H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; D208replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;E209 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; S210 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E211replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;V212 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q213 replacedwith D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; I214replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P215 replaced withD, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; M216replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; M217 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; Y218 replaced with D, E, H, K, R,N, Q, A, G, I, L, S, T, M, V, P, or C; Q219 replaced with D, E, H, K, R,A, G, I, L, S, T, M, V, F, W, Y, P, or C; Q220 replaced with D, E, H, K,R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; G221 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; E222 replaced with H, K, R, A, G, I, L, S,T, M, V, N, Q, F, W, Y, P, or C; F223 replaced with D, E, H, K, R, N, Q,A, G, I, L, S, T, M, V, P, or C; Y224 replaced with D, E, H, K, R, N, Q,A, G, I, L, S, T, M, V, P, or C; Y225 replaced with D, E, H, K, R, N, Q,A, G, I, L, S, T, M, V, P, or C; G226 replaced with D, E, H, K, R, N, Q,F, W, Y, P, or C; E227 replaced with H, K, R, A, G, I, L, S, T, M, V, N,Q, F, W, Y, P, or C; F228 replaced with D, E, H, K, R, N, Q, A, G, I, L,S, T, M, V, P, or C; S229 replaced with D, E, H, K, R, N, Q, F, W, Y, P,or C; D230 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,P, or C; G231 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S232replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N233 replaced withD, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; E234 replacedwith H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,P, or C; A235replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G236 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; G237 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; I238 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; Y239 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V,P, or C; Q240 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W,Y, P, or C; V241 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;L242 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E243 replacedwith H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; I244replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P245 replaced withD, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; Y246replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; E247replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;G248 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D249 replacedwith H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E250replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;I251 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S252 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; M253 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; M254 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; L255 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;V256 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L257 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; S258 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; R259 replaced with D, E, A, G, I, L, S, T,M, V, N, Q, F, W, Y, P, or C; Q260 replaced with D, E, H, K, R, A, G, I,L, S, T, M, V, F, W, Y, P, or C; E261 replaced with H, K, R, A, G, I, L,S, T, M, V, N, Q, F, W, Y, P, or C; V262 replaced with D, E, H, K, R, N,Q, F, W, Y, P, or C; P263 replaced with D, E, H, K, R, A, G, I, L, S, T,M, V, N, Q, F, W, Y, or C; L264 replaced with D, E, H, K, R, N, Q, F, W,Y, P, or C; A265 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;T266 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L267 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; E268 replaced with H, K, R,A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; P269 replaced with D, E,H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; L270 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; V271 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; K272 replaced with D, E, A, G, I, L, S, T, M, V,N, Q, F, W, Y, P, or C; A273 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; Q274 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W,Y, P, or C; L275 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;V276 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E277 replacedwith H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or 5 C; E278replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;W279 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;A280 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N281 replacedwith D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; S282replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;

[0107] V283 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K284replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K285replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Q286replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C;K287 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;V288 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E289 replacedwith H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V290replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y291 replaced withD, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; L292 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; P293 replaced with D, E, H, K, R,A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; R294 replaced with D, E, A,G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; F295 replaced with D, E, H,K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; T296 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; V297 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; E298 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q,F, W, Y, P, or C; Q299 replaced with D, E, H, K, R, A, G, I, L, S, T, M,V, F, W, Y, P, or C; E300 replaced with H, K, R, A, G, I, L, S, T, M, V,N, Q, F, W, Y, P, or C; I301 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; D302 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,Y, P, or C; L303 replaced 25 with D, E, H, K, R, N, Q, F, W, Y, P, or C;K304 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;D305 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; V306 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L307replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K308 replaced withD, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A309 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; L310 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; G311 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; I312 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T313replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E314 replaced withH, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; I315 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; F316 replaced with D, E, H,K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; I317 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; K318 replaced with D, E, A, G, I, L, S, T,M, V, N, Q, F, W, Y, P, or C; D319 replaced with H, K, R, A, G, I, L, S,T, M, V, N, Q, F, W, Y, P, or C; A320 replaced with D, E, H, K, R, N, Q,F, W, Y, P, or C; N321 replaced with D, E, H, K, R, A, G, I, L, S, T, M,V, F, W, Y, P, or C; L322 replaced with D, E, H, K, R, N, Q, F, W, Y, P,or C; T323 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G324replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L325 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; S326 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; D327 replaced with H, K, R, A, G, I, L, S, T, M,V, N, Q, F, W, Y, P, or C; N328 replaced with D, E, H, K, R, A, G, I, L,S, T, M, V, F, W, Y, P, or C; K329 replaced with D, E, A, G, I, L, S, T,M, V, N, Q, F, W, Y, P, or C; E330 replaced with H, K, R, A, G, 1, L, S,T, M, V, N, Q, F, W, Y, P, or C; I331 replaced with D, E, H, K, R, N, Q,F, W, Y, P, or C; F332 replaced with D, E, H, K, R, N, Q, A, G, I, L, S,T, M, V, P, or C; L333 replaced with D, E, H, K, R, N, Q, F, W, Y, P, orC; S334 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K335replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A336replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I337 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; H338 replaced with D, E, A, G, I,L, S, T, M, V, N, Q, F, W, Y, P, or C; K339 replaced with D, E, A, G, 1,L, S, T, M, V, N, Q, F, W, Y, P, or C; S340 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; F341 replaced with D, E, H, K, R, N, Q, A, G, I,L, S, T, M, V, P, or C; L342 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; E343 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,Y, P, or C; V344 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;N345 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, orC; E346 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,or C; E347 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,P, or C; G348 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S349replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E350 replaced withH, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A351 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; A352 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; A353 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; V354 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;S355 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G356 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; M357 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; I358 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; A359 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;I360 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S361 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; R362 replaced with D, E, A,G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; M363 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; A364 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; V365 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;L366 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y367 replacedwith D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; P368 replacedwith D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; Q369replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C;V370 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I371 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; V372 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; D373 replaced with H, K, R, A, G, I, L, S,T, M, V, N, Q, F, W, Y, P, or C; H374 replaced with D, E, A, G, I, L, S,T, M, V, N, Q, F, W, Y, P, or C; P375 replaced with D, E, H, K, R, A, G,I, L, S, T, M, V, N, Q, F, W, Y, or C; F376 replaced with D, E, H, K, R,N, Q, A, G, I, L, S, T, M, V, P, or C; F377 replaced with D, E, H, K, R,N, Q, A, G, I, L, S, T, M, V, P, or C; F378 replaced with D, E, H, K, R,N, Q, A, G, I, L, S, T, M, V, P, or C; L379 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; I380 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; R381 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y,P, or C; N382 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W,Y, P, or C; R383 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W,Y, P, or C; R384 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W,Y, P, or C; T385 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;G386 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T387 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; I388 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; L389 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; F390 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T,M, V, P, or C; M391 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;G392 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R393 replacedwith D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V394 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; M395 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; H396 replaced with D, E, A, G, I, L, S, T,M, V, N, Q, F, W, Y, P, or C; P397 replaced with D, E, H, K, R, A, G, I,L, S, T, M, V, N, Q, F, W, Y, or C; E398 replaced with H, K, R, A, G, I,L, S, T, M, V, N, Q, F, W, Y, P, or C; T399 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; M400 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; N401 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W,Y, P, or C; T402 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;S403 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G404 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; H405 replaced with ), E, A,G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; D406 replaced with H, K, R,A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; F407 replaced with D, E,H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; E408 replaced with H, K,R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E409 replaced with H,K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; and/or L410replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C of SEQ ID NO:2.

[0108] The resulting constructs can be routinely screened for activitiesor functions described throughout the specification and known in theart. Preferably, the resulting constructs have an increased and/ordecreased BAIT activity or function, while the remaining BAIT activitiesor functions are maintained. More preferably, the resulting constructshave more than one increased and/or decreased BAIT activity or function,while the remaining BAIT activities or functions are maintained.

[0109] Additionally, more than one amino acid (e.g., 2, 3, 4, 5, 6, 7,8, 9 and 10) can be replaced with the substituted amino acids asdescribed above (either conservative or nonconservative). Thesubstituted amino acids can occur in the full length, mature, orproprotein form of BAIT protein, as well as the N- and C-terminaldeletion mutants, having the general formula m-n.

[0110] A further embodiment of the invention relates to a polypeptidewhich comprises the amino acid sequence of a BAIT polypeptide having anamino acid sequence which contains at least one amino acid substitution,but not more than 50 amino acid substitutions, even more preferably, notmore than 40 amino acid substitutions, still more preferably, not morethan 30 amino acid substitutions, and still even more preferably, notmore than 20 amino acid substitutions. Of course, in order of ever-increasing preference, it is highly preferable for a polypeptide to havean amino acid sequence which comprises the amino acid sequence of a BAITpolypeptide, which contains at least one, but not more than 10, 9, 8, 7,6, 5, 4, 3, 2 or 1 amino acid substitutions. In specific embodiments,the number of additions, substitutions, and/or deletions in the aminoacid sequence of FIG. 1 or fragments thereof (e.g., the mature formand/or other fragments described herein), is 1-5, 5-10, 5-25, 5-50,10-50 or 50-150, conservative amino acid substitutions are preferable.

[0111] As described above, the BAIT polypeptide includes a reactivecenter loop (RCL) which interacts with its target proteinase. Shortpeptides (e.g., 8-30 residues) containing this loop sequence will bindto BAIT and convert it to a substrate for the target proteinase. Suchpeptides are therefore antagonists of BAIT and also form part of thepresent invention. Further, mutants of BAIT with enhanced function arealso provided by the invention, including: RCL replacements to increaseinhibitory activity with tPA, trypsin or thrombin; mutations thatincrease structural stability or clearance half-life; and mutationswhich enhance or block association with cofactors. One of ordinary skillwould appreciate that such mutants can be designed and tested using, forinstance, the methods described for other serpins in the referencescited in the section above on “Serpin Mechanism.”

[0112] The polypeptides of the present invention are preferably providedin an isolated form, and preferably are substantially purified. Arecombinantly produced version of the BAIT polypeptide can besubstantially purified by the method described in Osterwalder et al.,1996, supra.

[0113] The polypeptides of the present invention include the polypeptideencoded by the deposited cDNA including the leader, the maturepolypeptide encoded by the deposited cDNA minus the leader (i.e., themature protein), the polypeptide of FIG. 1 (SEQ ID NO:2) including theleader, the polypeptide of FIG. 1 (SEQ ID NO:2) minus the leader, aswell as polypeptides which have at least 90% similarity, more preferablyat least 95% similarity, and still more preferably at least 96%, 97%,98% or 99% similarity to those described above. Further polypeptides ofthe present invention include polypeptides at least 80% identical, morepreferably at least 90% or 95% identical, still more preferably at least96%, 97%, 98% or 99% identical to the polypeptide encoded by thedeposited cDNA, to the polypeptide of FIG. 1 (SEQ ID NO:2), and alsoinclude portions of such polypeptides with at least 30 amino acids andmore preferably at least 50 amino acids.

[0114] By “% similarity” for two polypeptides is intended a similarityscore produced by comparing the amino acid sequences of the twopolypeptides using the Bestfit program (Wisconsin Sequence AnalysisPackage, Version 8 for Unix, Genetics Computer Group, UniversityResearch Park, 575 Science Drive, Madison, Wis. 53711) and the defaultsettings for determining similarity. Bestfit uses the local homologyalgorithm of Smith and Waterman (Advances in Applied Mathematics2:482-489, 1981) to find the best segment of similarity between twosequences.

[0115] By a polypeptide having an amino acid sequence at least, forexample, 95% “identical” to a reference amino acid sequence of a BAITpolypeptide is intended that the amino acid sequence of the polypeptideis identical to the reference sequence except that the polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the reference amino acid of the BAIT polypeptide. Inother words, to obtain a polypeptide having an amino acid sequence atleast 95% identical to a reference amino acid sequence, up to 5% of theamino acid residues in the reference sequence may be deleted orsubstituted with another amino acid, or a number of amino acids up to 5%of the total amino acid residues in the reference sequence may beinserted into the reference sequence. These alterations of the referencesequence may occur at the amino or carboxy terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more contiguous groups within thereference sequence.

[0116] As a practical matter, whether any particular polypeptide is atleast 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, theamino acid sequence shown in FIG. 1 (SEQ ID NO:2) or to the amino acidsequence encoded by deposited cDNA clone can be determinedconventionally using known computer programs such the Bestfit program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, 575 Science Drive, Madison,Wis. 53711). When using Bestfit or any other sequence alignment programto determine whether a particular sequence is, for instance, 95%identical to a reference sequence according to the present invention,the parameters are set, of course, such that the percentage of identityis calculated over the full length of the reference amino acid sequenceand that gaps in homology of up to 5% of the total number of amino acidresidues in the reference sequence are allowed.

[0117] The polypeptide of the present invention could be used as amolecular weight marker on SDS-PAGE gels or on molecular sieve gelfiltration columns using methods well known to those of skill in theart. As described in detail below, the polypeptides of the presentinvention can also be used to raise polyclonal and monoclonalantibodies, which are useful in assays for detecting BAIT proteinexpression as described below or as antagonists capable of enhancing orinhibiting BAIT protein function. Further, such polypeptides can be usedin the yeast two-hybrid system to “capture” BAIT protein bindingproteins which are candidate target proteins for BAIT inhibition,according to the present invention. The yeast two hybrid system isdescribed in Fields and Song, Nature 340:245-246 (1989).

[0118] Additional preferred polypeptide fragments comprise, oralternatively consist of, the amino acid sequence of residues: M-1 toM-15; A-2 to A-16; F-3 to T-17; L-4 to G-18; G-5 to A-19; L-6 to T-20;F-7 to F-21; S-8 to P-22; L-9 to E-23; L-10 to E-24; V-11 to A-25; L-12to 1-26; Q-13 to A-27; S-14 to D-28; M-15 to L-29; A-16 to S-30;T-17 toV-31; G-18 to N-32; A-19 to M-33; T-20 to Y-34; F-21 to N-35; P-22 toR-36; E-23 to L-37; E-24 to R-38; A-25 to A-39; 1-26 to T-40; A-27 toG-41; D-28 to E-42; L-29 to D-43; S-30 to E-44; V-31 to N-45; N-32 to1-46; M-33 to L-47; Y-34 to F-48; N- 35 to S-49; R-36 to P-50; L-37 toL-51; R-38 to S-52;A-39 to I-53; T-40 to A-54; G-41 to L-55; E-42 toA-56; D-43 to M-57; E-44 to G-58; N-45 to M-59; 1-46 to M-60; L-47 toE-61; F-48 to L-62; S-49 to G-63; P-50 to A-64; L-51 to Q-65; S-52 toG-66; I-53 to S-67; A-54 to T-68; L-55 to Q-69; A-56 to K-70; M-57 toE-71; G-58 to I-72; M-59 to R-73; M-60 to H-74; E-61 to S-75; L-62 toM-76; G-63 to G-77; A-64 to Y-78; Q-65 to D-79; G-66 to S-80; S-67 toL-81; T-68 to K-82; Q-69 to N-83; K-70 to G-84; E-71 to E-85; 1-72 toE-86; R-73 to F-87; H-74 to S-88; S-75 to F-89; M-76 to L-90; G-77 toK-91; Y-78 to E-92; D-79 to F-93; S-80 to S-94; L-81 to N-95; K-82 toM-96; N-83 to V-97; G-84 to T-98; E-85 to A-99; E-86 to K-100; F-87 toE-101; S-88 to S-102; F-89 to Q-103; L-90 to Y-104; K-91 to V-105; E-92to M-106; F-93 to K-107; S-94 to I-108; N-95 to A-109; M-96 to N-110;V-97 to S-111; T-98 to L-112; A-99 to F-113; K-100 to V-114; E-101 toQ-115; S-102 to N-116; Q-103 to G-117; Y-104 to F-118; V-105 to H-119;M-106 to V-120; K-107 to N-121; I-108 to E-122; A-109 to E-123; N-110 toF-124; S-Ill to L-125; L-112 to Q-126; F-113 to M-127; V-114 to M-128;Q-115 to K-129; N-116 to K-130; G-117 to Y-131; F-118 to F-132; H-119 toN-133; V-120 to A-134; N-121 to A-135; E-122 to V-136; E-123 to N-137;F-124 to H-138; L-125 to V-139; Q-126 to D-140; M-127 to F-141; M-128 toS-142; K-129 to Q-143; K-130 to N-144; Y-131 to V-145; F-132 to A-146;N-133 to V-147; A-134 to A-148; A-135 to N-149; V-136 to Y-150; N-137 to1-151; H-138 to N-152; V-139 to K-153; D-140 to W-154; F-141 to V-155;S-142 to E-156; Q-143 to N-157; N-144 to N-158; V-145 to T-159; A-146 toN-160; V-147 to N -161; A-148 to L-162; N-149 to V-163; Y-150 to K-164;1-151 to D-165; N-152 to L-166; K-153 to V-167; W-154 to S-168; V-155 toP-169; E-156 to R-170; N-157 to D-171; N-158 to F-172; T-159 to D-173;N-160 to A-174; N-161 to A-175; L-162 to T-176; V-163 to Y-177; K-164 toL-178; D-165 to A-179; L-166 to L-180; V-167 to I-181; S-168 to N-182;P-169 to A-183; R-170 to V-184; D-171 to Y-185; F-172 to F-186; D-173 toK-187; A-174 to G-188; A-175 to N-189; T-176 to W-190; Y-177 to K-191;L-178 to S-192; A-179 to Q-193; L-180 to F-194; I-181 to R-195; N-182 toP-196; A-183 to E-197; V-184 to N-198; Y-185 to T-199; F-186 to R-200;K-187 to T-201; G-188 to F-202; N-189 to S-203; W-190 to F-204; K-191 toT-205; S-192 to K-206; Q-193 to D-207; F-194 to D-208; R-195 to E-209;P-196 to S-210; E-197 to E-211; N-198 to V-212; T-199 to Q-213; R-200 toI-214; T-201 to P-215; F-202 to M-216; S-203 to M-217; F-204 to Y-218;T-205 to Q-219; K-206 to Q-220; D-207 to G-221; D-208 to E-222; E-209 toF-223; S-210 to Y-224; E-211 to Y-225; V-212 to G-226; Q-213 to E-227;1-214 to F-228; P-215 to S-229; M-216 to D-230; M-217 to G-231; Y-218 toS-232; Q-219 to N-233; Q-220 to E- 234; G-221 to A-235; E-222 to G-236;F-223 to G-237; Y-224 to 1-238; Y-225 to Y-239; G-226 to Q-240; E-227 toV-241; F-228 to L-242; S-229 to E-243; D-230 to I-244; G-231 to P-245;S-232 to Y-246; N-233 to E-247; E-234 to G-248; A-235 to D-249; G-236 toE-250; G-237 to I-251; 1-238 to S-252; Y-239 to M-253; Q-240 to M-254;V-241 to L-255; L-242 to V-256; E-243 to L-257; 1-244 to S-258; P-245 toR-259; Y-246 to Q-260; E-247 to E-261; G-248 to V-262; D-249 to P-263;E-250 to L-264; I-251 to A-265; S-252 to T-266; M-253 to L-267; M-254 toE-268; L-255 to P-269; V-256 to L-270; L-257 to V-271; S-258 to K-272;R-259 to A-273; Q-260 to Q-274; E-261 to L-275; V-262 to V-276; P-263 toE-277; L-264 to E-278; A-265 to W-279; T-266 to A-280; L-267 to N-281;E-268 to S-282; P-269 to V-283; L-270 to K-284; V-271 to K-285; K-272 toQ-286; A-273 to K-287; Q-274 to V-288; L-275 to E-289; V-276 to V-290;E-277 to Y-291; E-278 to L-292; W-279 to P-293; A-280 to R-294; N-281 toF-295; S-282 to T-296; V-283 to V-297; K-284 to E-298; K-285 to Q-299;Q-286 to E-300; K-287 to 1-301; V-288 to D-302; E-289 to L-303; V-290 toK-304; Y-291 to D-305; L-292 to V-306; P-293 to L-307; R-294 to K-308;F-295 to A-309; T-296 to L-310; V-297 to G-311; E-298 to 1-312; Q-299 toT-313; E-300 to E-314; 1-301 to 1-315; D-302 to F-316; L-303 to 1-317;K-304 to K-318; D-305 to D-319; V-306 to A-320; L-307 to N-321; K-308 toL-322; A-309 to T-323; L-310 to G-324; G-311 to L-325; 1-312 to S-326;T-313 to D-327; E-314 to N-328; I-315 to K-329; F-316 to E-330; 1-317 to1-331; K-318 to F-332; D-319 to L-333; A-320 to S-334; N-321 to K-335;L-322 to A-336; T-323 to 1-337; G-324 to H-338; L-325 to K-339; S-326 toS-340; D-327 to F-341; N-328 to L-342; K-329 to E-343; E-330 to V-344;1-331 to N-345; F-332 to E-346; L-333 to E-347; S-334 to G-348; K-335 toS-349; A-336 to E-350; 1-337 to A-351; H-338 to A-352; K-339 to A-353;S-340 to V-354; F-341 to S-355; L-342 to G-356; E-343 to M-357; V-344 to1-358; N-345 to A-359; E-346 to I-360; E-347 to S-361; G-348 to R-362;S-349 to M-363; E-350 to A-364; A-351 to V-365; A-352 to L-366; A-353 toY-367; V-354 to P-368; S-355 to Q-369; G-356 to V-370; M-357 to 1-371;1-358 to V-372; A-359 to D-373; 1-360 to H-374; S-361 to P-375; R-362 toF-376; M-363 to F-377; A-364 to F-378; V-365 to L-379; L-366 to I-380;Y-367 to R-381; P-368 to N-382; Q-369 to R-383; V-370 to R-384; 1-371 toT-385; V-372 to G-386; D-373 to T-387; H-374 to I-388; P-375 to L-389;F-376 to F-390; F-377 to M-391; F-378 to G-392; L-379 to R-393; 1-380 toV-394; R-381 to M-395; N-382 to H-396; R-383 to P-397; R-384 to E-398;T-385 to T-399; G-386 to M-400; T-387 to N-401; 1-388 to T-402; L-389 toS-403; F-390 to G-404; M-391 to H-405; G-392 to D-406; R-393 to F-407;V-394 to E-408; M-395 to E-409; and/or H-396 to L-410 of SEQ ID NO:2.These polypeptide fragments may retain the biological activity of theBAIT polypeptides of the invention and/or may be useful to generate orscreen for antibodies, as described further below. Polynucleotidesencoding these polypeptide fragments are also encompassed by theinvention.

[0119] Epitopes and Antibodies

[0120] The present invention encompasses polypeptides comprising, oralternatively consisting of, an epitope of the polypeptide having anamino acid sequence of SEQ ID NO:2, or an epitope of the polypeptidesequence encoded by a polynucleotide sequence contained in ATCC depositNo. 97722 or encoded by a polynucleotide that hybridizes to thecomplement of the sequence of SEQ ID NO:1 or contained in ATCC depositNo. 97722 under stringent hybridization conditions or lower stringencyhybridization conditions as defined supra. The present invention furtherencompasses polynucleotide sequences encoding an epitope of apolypeptide sequence of the invention (such as, for example, thesequence disclosed in SEQ ID NO:2), polynucleotide sequences of thecomplementary strand of a polynucleotide sequence encoding an epitope ofthe invention, and polynucleotide sequences which hybridize to thecomplementary strand under stringent hybridization conditions or lowerstringency hybridization conditions defined supra.

[0121] The term “epitopes,” as used herein, refers to portions of apolypeptide having antigenic or immunogenic activity in an animal,preferably a mammal, and most preferably in a human. In a preferredembodiment, the present invention encompasses a polypeptide comprisingan epitope, as well as the polynucleotide encoding this polypeptide. An“immunogenic epitope,” as used herein, is defined as a portion of aprotein that elicits an antibody response in an animal, as determined byany method known in the art, for example, by the methods for generatingantibodies described infra. (See, for example, Geysen et al., Proc.Natl. Acad. Sci. USA 81:3998-4002 (1983)). The term “antigenic epitope,”as used herein, is defined as a portion of a protein to which anantibody can immunospecifically bind its antigen as determined by anymethod well known in the art, for example, by the immunoassays describedherein. Immunospecific binding excludes non-specific binding but doesnot necessarily exclude cross-reactivity with other antigens. Antigenicepitopes need not necessarily be immunogenic.

[0122] Fragments which function as epitopes may be produced by anyconventional means. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA82:5131-5135 (1985), further described in U.S. Pat. No. 4,631,211).

[0123] In the present invention, antigenic epitopes preferably contain asequence of at least 4, at least 5, at least 6, at least 7, morepreferably at least 8, at least 9, at least 10, at least 11, at least12, at least 13, at least 14, at least 15, at least 20, at least 25, atleast 30, at least 40, at least 50, and, most preferably, between about15 to about 30 amino acids. Preferred polypeptides comprisingimmunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acidresidues in length. Additional non-exclusive preferred antigenicepitopes include the antigenic epitopes disclosed herein, as well asportions thereof. Specifically prefered are epitopes comprising, orconsisting of: a polypeptide comprising amino acid residues from aboutVal 31 to about Leu 47 (SEQ ID NO:2); a polypeptide comprising aminoacid residues from about Leu 62 to about Ser 88 (SEQ ID NO:2); apolypeptide comprising amino acid residues from about Val 155 to aboutAla 175 (SEQ ID NO:2); a polypeptide comprising amino acid residues fromabout Phe 186 to about Pro 215 (SEQ ID NO:2); a polypeptide comprisingamino acid residues from about Tyr 225 to about Ile 239 (SEQ ID NO:2); apolypeptide comprising amino acid residues from about Leu 243 to aboutLeu 255 (SEQ ID NO:2); a polypeptide comprising amino acid residues fromabout Arg 380 to about Gly 386 (SEQ ID NO:2); and a polypeptidecomprising amino acid residues from about Met 395 to about Leu 410. (SEQID NO:2). Antigenic epitopes are useful, for example, to raiseantibodies, including monoclonal antibodies, that specifically bind theepitope. Preferred antigenic epitopes include the antigenic epitopesdisclosed herein, as well as any combination of two, three, four, fiveor more of these antigenic epitopes. Antigenic epitopes can be used asthe target molecules in immunoassays. (See, for instance, Wilson et al.,Cell 37:767-778 (1984); Sutcliffe et al., Science 219:660-666 (1983)).

[0124] Similarly, immunogenic epitopes can be used, for example, toinduce antibodies according to methods well known in the art. (See, forinstance, Sutcliffe et al., supra; Wilson et al., supra; Chow et al.,Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al., J. Gen. Virol.66:2347-2354 (1985). Preferred immunogenic epitopes include theimmunogenic epitopes disclosed herein, as well as any combination oftwo, three, four, five or more of these immunogenic epitopes. Thepolypeptides comprising one or more immunogenic epitopes may bepresented for eliciting an antibody response together with a carrierprotein, such as an albumin, to an animal system (such as rabbit ormouse), or, if the polypeptide is of sufficient length (at least about25 amino acids), the polypeptide may be presented without a carrier.However, immunogenic epitopes comprising as few as 8 to 10 amino acidshave been shown to be sufficient to raise antibodies capable of bindingto, at the very least, linear epitopes in a denatured polypeptide (e.g.,in Western blotting).

[0125] Epitope-bearing polypeptides of the present invention may be usedto induce antibodies according to methods well known in the artincluding, but not limited to, in vivo immunization, in vitroimmunization, and phage display methods. See, e.g., Sutcliffe et al.,supra; Wilson et al., supra, and Bittle et al., J. Gen. Virol.,66:2347-2354 (1985). If in vivo immunization is used, animals may beimmunized with free peptide; however, anti-peptide antibody titer may beboosted by coupling the peptide to a macromolecular carrier, such askeyhole limpet hemacyanin (KLH) or tetanus toxoid. For instance,peptides containing cysteine residues may be coupled to a carrier usinga linker such as maleimidobenzoyl-N-hydroxysuccinimide ester (MBS),while other peptides may be coupled to carriers using a more generallinking agent such as glutaraldehyde. Animals such as rabbits, rats andmice are immunized with either free or carrier-coupled peptides, forinstance, by intraperitoneal and/or intradermal injection of emulsionscontaining about 100 μg of peptide or carrier protein and Freund'sadjuvant or any other adjuvant known for stimulating an immune response.Several booster injections may be needed, for instance, at intervals ofabout two weeks, to provide a useful titer of anti-peptide antibodywhich can be detected, for example, by ELISA assay using free peptideadsorbed to a solid surface. The titer of anti-peptide antibodies inserum from an immunized animal may be increased by selection ofanti-peptide antibodies, for instance, by adsorption to the peptide on asolid support and elution of the selected antibodies according tomethods well known in the art.

[0126] As one of skill in the art will appreciate, and as discussedabove, the polypeptides of the present invention comprising animmunogenic or antigenic epitope can be fused to other polypeptidesequences. For example, the polypeptides of the present invention may befused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM),or portions thereof (CH1, CH2, CH3, or any combination thereof andportions thereof) resulting in chimeric polypeptides. Such fusionproteins may facilitate purification and may increase half-life in vivo.This has been shown for chimeric proteins consisting of the first twodomains of the human CD4-polypeptide and various domains of the constantregions of the heavy or light chains of mammalian immunoglobulins. See,e.g., EP 394,827; Traunecker et al., Nature, 331:84-86 (1988). Enhanceddelivery of an antigen across the epithelial barrier to the immunesystem has been demonstrated for antigens (e.g., insulin) conjugated toan FcRn binding partner such as IgG or Fc fragments (see, e.g., PCTPublications WO 96/22024 and WO 99/04813). IgG Fusion proteins that havea disulfide-linked dimeric structure due to the IgG portion desulfidebonds have also been found to be more efficient in binding andneutralizing other molecules than monomeric polypeptides or fragmentsthereof alone. See, e.g., Fountoulakis et al., J. Biochem.,270:3958-3964 (1995). Nucleic acids encoding the above epitopes can alsobe recombined with a gene of interest as an epitope tag (e.g., thehemagglutinin (“HA”) tag or flag tag) to aid in detection andpurification of the expressed polypeptide. For example, a systemdescribed by Janknecht et al. allows for the ready purification ofnon-denatured fusion proteins expressed in human cell lines (Janknechtet al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-897). In this system,the gene of interest is subcloned into a vaccinia recombination plasmidsuch that the open reading frame of the gene is translationally fused toan amino-terminal tag onsisting of six histidine residues. The tagserves as a matrix binding domain for the fusion protein. Extracts fromcells infected with the recombinant vaccinia virus are loaded onto Ni2+nitriloacetic acid-agarose column and histidine-tagged proteins can beselectively eluted with imidazole-containing buffers.

[0127] Additional fusion proteins of the invention may be generatedthrough the techniques of gene-shuffling, motif-shuffling,exon-shuffling, and/or codon-shuffling (collectively referred to as “DNAshuffling”). DNA shuffling may be employed to modulate the activities ofpolypeptides of the invention, such methods can be used to generatepolypeptides with altered activity, as well as agonists and antagonistsof the polypeptides. See, generally, U.S. Pat. Nos. 5,605,793;5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et al., Curr.Opinion Biotechnol. 8:724-33 (1997); Harayama, Trends Biotechnol.16(2):76-82 (1998); Hansson, et al., J. Mol. Biol. 287:265-76 (1999);and Lorenzo and Blasco, Biotechniques 24(2):308-13 (1998) (each of thesepatents and publications are hereby incorporated by reference in itsentirety). In one embodiment, alteration of polynucleotidescorresponding to SEQ ID NO:1 and the polypeptides encoded by thesepolynucleotides may be achieved by DNA shuffling. DNA shuffling involvesthe assembly of two or more DNA segments by homologous or site-specificrecombination to generate variation in the polynucleotide sequence. Inanother embodiment, polynucleotides of the invention, or the encodedpolypeptides, may be altered by being subjected to random mutagenesis byerror-prone PCR, random nucleotide insertion or other methods prior torecombination. In another embodiment, one or more components, motifs,sections, parts, domains, fragments, etc., of a polynucleotide encodinga polypeptide of the invention may be recombined with one or morecomponents, motifs, sections, parts, domains, fragments, etc. of one ormore heterologous molecules.

[0128] Antibodies

[0129] Further polypeptides of the invention relate to antibodies andT-cell antigen receptors (TCR) which immunospecifically bind apolypeptide, polypeptide fragment, or variant of SEQ ID NO:2, and/or anepitope, of the present invention (as determined by immunoassays wellknown in the art for assaying specific antibody-antigen binding).Antibodies of the invention include, but are not limited to, polyclonal,monoclonal, multispecific, human, humanized or chimeric antibodies,single chain antibodies, Fab fragments, F(ab′) fragments, fragmentsproduced by a Fab expression library, anti-idiotypic (anti-Id)antibodies (including, e.g., anti-Id antibodies to antibodies of theinvention), and epitope-binding fragments of any of the above. The term“antibody,” as used herein, refers to immunoglobulin molecules andimmunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site that immunospecificallybinds an antigen. The immunoglobulin molecules of the invention can beof any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1,IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.

[0130] Most preferably the antibodies are human antigen-binding antibodyfragments of the present invention and include, but are not limited to,Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chainantibodies, disulfide-linked Fvs (sdFv) and fragments comprising eithera VL or VH domain. Antigen-binding antibody fragments, includingsingle-chain antibodies, may comprise the variable region(s) alone or incombination with the entirety or a portion of the following: hingeregion, CH1, CH2, and CH3 domains. Also included in the invention areantigen-binding fragments also comprising any combination of variableregion(s) with a hinge region, CH1, CH2, and CH3 domains. The antibodiesof the invention may be from any animal origin including birds andmammals. Preferably, the antibodies are human, murine (e.g., mouse andrat), donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken.As used herein, “human” antibodies include antibodies having the aminoacid sequence of a human immunoglobulin and include antibodies isolatedfrom human immunoglobulin libraries or from animals transgenic for oneor more human immunoglobulin and that do not express endogenousimmunoglobulins, as described infra and, for example in, U.S. Pat. No.5,939,598 by Kucherlapati et al.

[0131] The antibodies of the present invention may be monospecific,bispecific, trispecific or of greater multispecificity. Multispecificantibodies may be specific for different epitopes of a polypeptide ofthe present invention or may be specific for both a polypeptide of thepresent invention as well as for a heterologous epitope, such as aheterologous polypeptide or solid support material. See, e.g., PCTpublications WO 93/17715; WO 92/08802; WO 91/00360; WO 92105793; Tutt,et al., J. Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893;4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol.148:1547-1553 (1992).

[0132] Antibodies of the present invention may be described or specifiedin terms of the epitope(s) or portion(s) of a polypeptide of the presentinvention which they recognize or specifically bind. The epitope(s) orpolypeptide portion(s) may be specified as described herein, e.g., byN-terminal and C-terminal positions, by size in contiguous amino acidresidues, or listed in the Tables and Figures. Antibodies whichspecifically bind any epitope or polypeptide of the present inventionmay also be excluded. Therefore, the present invention includesantibodies that specifically bind polypeptides of the present invention,and allows for the exclusion of the same. Antibodies of the presentinvention may also be described or specified in terms of theircross-reactivity. Antibodies that do not bind any other analog,ortholog, or homolog of a polypeptide of the present invention areincluded. Antibodies that bind polypeptides with at least 95%, at least90%, at least 85%, at least 80%, at least 75%, at least 70%, at least65%, at least 60%, at least 55%, and at least 50% identity (ascalculated using methods known in the art and described herein) to apolypeptide of the present invention are also included in the presentinvention. In specific embodiments, antibodies of the present inventioncross-react with murine, rat and/or rabbit homologs of human proteinsand the corresponding epitopes thereof. Antibodies that do not bindpolypeptides with less than 95%, less than 90%, less than 85%, less than80%, less than 75%, less than 70%, less than 65%, less than 60%, lessthan 55%, and less than 50% identity (as calculated using methods knownin the art and described herein) to a polypeptide of the presentinvention are also included in the present invention. In a specificembodiment, the above-described cross-reactivity is with respect to anysingle specific antigenic or immunogenic polypeptide, or combination(s)of 2, 3, 4, 5, or more of the specific antigenic and/or immunogenicpolypeptides disclosed herein. Further included in the present inventionare antibodies which bind polypeptides encoded by polynucleotides whichhybridize to a polynucleotide of the present invention under stringenthybridization conditions (as described herein). Antibodies of thepresent invention may also be described or specified in terms of theirbinding affinity to a polypeptide of the invention. Preferred bindingaffinities include those with a dissociation constant or Kd less than5×10⁻² M, 10⁻² M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, 10⁻⁵ M,5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷ M, 10⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M,5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M,10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, or 10⁻¹⁵ M.

[0133] The invention also provides antibodies that competitively inhibitbinding of an antibody to an epitope of the invention as determined byany method known in the art for determining competitive binding, forexample, the immunoassays described herein. In preferred embodiments,the antibody competitively inhibits binding to the epitope by at least95%, at least 90%, at least 85%, at least 80%, at least 75%, at least70%, at least 60%, or at least 50%.

[0134] Antibodies of the present invention may act as agonists orantagonists of the polypeptides of the present invention. For example,the present invention includes antibodies which disrupt thereceptor/ligand interactions with the polypeptides of the inventioneither partially or fully. Preferably, antibodies of the presentinvention bind an antigenic epitope disclosed herein, or a portionthereof. The invention features both receptor-specific antibodies andligand-specific antibodies. The invention also featuresreceptor-specific antibodies which do not prevent ligand binding butprevent receptor activation. Receptor activation (i.e., signaling) maybe determined by techniques described herein or otherwise known in theart. For example, receptor activation can be determined by detecting thephosphorylation (e.g., tyrosine or serine/threonine) of the receptor orits substrate by immunoprecipitation followed by western blot analysis(for example, as described supra). In specific embodiments, antibodiesare provided that inhibit ligand activity or receptor activity by atleast 95%, at least 90%, at least 85%, at least 80%, at least 75%, atleast 70%, at least 60%, or at least 50% of the activity in absence ofthe antibody.

[0135] The invention also features receptor-specific antibodies whichboth prevent ligand binding and receptor activation as well asantibodies that recognize the receptor-ligand complex, and, preferably,do not specifically recognize the unbound receptor or the unboundligand. Likewise, included in the invention are neutralizing antibodieswhich bind the ligand and prevent binding of the ligand to the receptor,as well as antibodies which bind the ligand, thereby preventing receptoractivation, but do not prevent the ligand from binding the receptor.Further included in the invention are antibodies which activate thereceptor. These antibodies may act as receptor agonists, i.e.,potentiate or activate either all or a subset of the biologicalactivities of the ligand-mediated receptor activation, for example, byinducing dimerization of the receptor. The antibodies may be specifiedas agonists, antagonists or inverse agonists for biological activitiescomprising the specific biological activities of the peptides of theinvention disclosed herein. The above antibody agonists can be madeusing methods known in the art. See, e.g., PCT publication WO 96/40281;U.S. Pat. No. 5,811,097; Deng et al., Blood 92(6):1981-1988 (1998); Chenet al., Cancer Res. 58(16):3668-3678 (1998); Harrop et al., J. Immunol.161(4):1786-1794 (1998); Zhu et al., Cancer Res. 58(15):3209-3214(1998); Yoon et al., J. Immunol. 160(7):3170-3179 (1998); Prat et al.,J. Cell. Sci. 111(Pt2):237-247 (1998); Pitard et al., J. Immunol.Methods 205(2):177-190 (1997); Liautard et al., Cytokine 9(4):233-241(1997); Carlson et al., J. Biol. Chem. 272(17):11295-11301 (1997);Taryman et al., Neuron 14(4):755-762 (1995); Muller et al., Structure6(9):1153-1167 (1998); Bartunek et al., Cytokine 8(1):14-20 (1996)(which are all incorporated by reference herein in their entireties).

[0136] Antibodies of the present invention may be used, for example, butnot limited to, to purify, detect, and target the polypeptides of thepresent invention, including both in vitro and in vivo diagnostic andtherapeutic methods. For example, the antibodies have use inimmunoassays for qualitatively and quantitatively measuring levels ofthe polypeptides of the present invention in biological samples. See,e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold SpringHarbor Laboratory Press, 2nd ed. 1988) (incorporated by reference hereinin its entirety).

[0137] As discussed in more detail below, the antibodies of the presentinvention may be used either alone or in combination with othercompositions. The antibodies may further be recombinantly fused to aheterologous polypeptide at the N- or C-terminus or chemicallyconjugated (including covalently and non-covalently conjugations) topolypeptides or other compositions. For example, antibodies of thepresent invention may be recombinantly fused or conjugated to moleculesuseful as labels in detection assays and effector molecules such asheterologous polypeptides, drugs, radionuclides, or toxins. See, e.g.,PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No.5,314,995; and EP 396,387.

[0138] The antibodies of the invention include derivatives that aremodified, i.e., by the covalent attachment of any type of molecule tothe antibody such that covalent attachment does not prevent the antibodyfrom generating an anti-idiotypic response. For example, but not by wayof limitation, the antibody derivatives include antibodies that havebeen modified, e.g., by glycosylation, acetylation, pegylation,phosphylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Additionally, the derivative may contain one or more non-classicalamino acids.

[0139] The antibodies of the present invention may be generated by anysuitable method known in the art. Polyclonal antibodies to anantigen-of- interest can be produced by various procedures well known inthe art. For example, a polypeptide of the invention can be administeredto various host animals including, but not limited to, rabbits, mice,rats, etc. to induce the production of sera containing polyclonalantibodies specific for the antigen. Various adjuvants may be used toincrease the immunological response, depending on the host species, andinclude but are not limited to, Freund's (complete and incomplete),mineral gels such as aluminum hydroxide, surface active substances suchas lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and corynebacteriumparvum. Such adjuvants are also well known in the art.

[0140] Monoclonal antibodies can be prepared using a wide variety oftechniques known in the art including the use of hybridoma, recombinant,and phage display technologies, or a combination thereof. For example,monoclonal antibodies can be produced using hybridoma techniquesincluding those known in the art and taught, for example, in Harlow etal., Antibodies: A Laboratory Manual, (Cold Spring Harbor LaboratoryPress, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies andT-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (said referencesincorporated by reference in their entireties). The term “monoclonalantibody” as used herein is not limited to antibodies produced throughhybridoma technology. The term “monoclonal antibody” refers to anantibody that is derived from a single clone, including any eukaryotic,prokaryotic, or phage clone, and not the method by which it is produced.

[0141] Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art and arediscussed in detail in the Examples. In a non-limiting example, mice canbe immunized with a polypeptide of the invention or a cell expressingsuch peptide. Once an immune response is detected, e.g., antibodiesspecific for the antigen are detected in the mouse serum, the mousespleen is harvested and splenocytes isolated. The splenocytes are thenfused by well known techniques to any suitable myeloma cells, forexample cells from cell line SP20 available from the ATCC. Hybridomasare selected and cloned by limited dilution. The hybridoma clones arethen assayed by methods known in the art for cells that secreteantibodies capable of binding a polypeptide of the invention. Ascitesfluid, which generally contains high levels of antibodies, can begenerated by immunizing mice with positive hybridoma clones.

[0142] Accordingly, the present invention provides methods of generatingmonoclonal antibodies as well as antibodies produced by the methodcomprising culturing a hybridoma cell secreting an antibody of theinvention wherein, preferably, the hybridoma is generated by fusingsplenocytes isolated from a mouse immunized with an antigen of theinvention with myeloma cells and then screening the hybridomas resultingfrom the fusion for hybridoma clones that secrete an antibody able tobind a polypeptide of the invention.

[0143] Antibody fragments which recognize specific epitopes may begenerated by known techniques. For example, Fab and F(ab′)2 fragments ofthe invention may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain thevariable region, the light chain constant region and the CHI domain ofthe heavy chain.

[0144] For example, the antibodies of the present invention can also begenerated using various phage display methods known in the art. In phagedisplay methods, functional antibody domains are displayed on thesurface of phage particles which carry the polynucleotide sequencesencoding them. In a particular embodiment, such phage can be utilized todisplay antigen binding domains expressed from a repertoire orcombinatorial antibody library (e.g., human or murine). Phage expressingan antigen binding domain that binds the antigen of interest can beselected or identified with antigen, e.g., using labeled antigen orantigen bound or captured to a solid surface or bead. Phage used inthese methods are typically filamentous phage including fd and M13binding domains expressed from phage with Fab, Fv or disulfidestabilized Fv antibody domains recombinantly fused to either the phagegene III or gene VIII protein. Examples of phage display methods thatcan be used to make the antibodies of the present invention includethose disclosed in Brinkman et al., J. Imrnunol. Methods 182:41-50(1995); Ames et al., J. Immunol. Methods 184:177-186 (1995);Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al.,Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280(1994); PCT application No. PCT/GB91/01134; PCT publications WO90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409;5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047;

[0145]5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743and 5,969,108; each of which is incorporated herein by reference in itsentirety.

[0146] As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′ and F(ab′)2 fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869(1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al.,Science 240:1041-1043 (1988) (said references incorporated by referencein their entireties).

[0147] Examples of techniques which can be used to produce single-chainFvs and antibodies include those described in U.S. Pat. Nos. 4,946,778and 5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991);Shu et al., PNAS 90:7995-7999 (1993); and Skerra et al., Science240:1038-1040 (1988). For some uses, including in vivo use of antibodiesin humans and in vitro detection assays, it may be preferable to usechimeric, humanized, or human antibodies. A chimeric antibody is amolecule in which different portions of the antibody are derived fromdifferent animal species, such as antibodies having a variable regionderived from a murine monoclonal antibody and a human immunoglobulinconstant region. Methods for producing chimeric antibodies are known inthe art. See e.g., Morrison, Science 229:1202 (1985); Oi et al.,BioTechniques 4:214 (1986); Gillies et al., (1989) J. Immunol. Methods125:191-202; U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816397, whichare incorporated herein by reference in their entirety. Humanizedantibodies are antibody molecules from non-human species antibody thatbinds the desired antigen having one or more complementarity determiningregions (CDRs) from the non-human species and a framework regions from ahuman immunoglobulin molecule. Often, framework residues in the humanframework regions will be substituted with the corresponding residuefrom the CDR donor antibody to alter, preferably improve, antigenbinding. These framework substitutions are identified by methods wellknown in the art, e.g., by modeling of the interactions of the CDR andframework residues to identify framework residues important for antigenbinding and sequence comparison to identify unusual framework residuesat particular positions. (See, e.g., Queen et al., U.S. Pat. No.5,585,089; Riechmann et al., Nature 332:323 (1988), which areincorporated herein by reference in their entireties.) Antibodies can behumanized using a variety of techniques known in the art including, forexample, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S.Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing(EP 592,106; EP 519,596; Padlan, Molecular Immunology28(4/5):489-498-(1991); Studnicka et al., Protein Engineering7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994)), and chainshuffling (U.S. Pat. No. 5,565,332).

[0148] Completely human antibodies are particularly desirable fortherapeutic treatment of human patients. Human antibodies can be made bya variety of methods known in the art including phage display methodsdescribed above using antibody libraries derived from humanimmunoglobulin sequences. See also, U.S. Pat. Nos. 4,444,887 and4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893,WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which isincorporated herein by reference in its entirety.

[0149] Human antibodies can also be produced using transgenic mice whichare incapable of expressing functional endogenous immunoglobulins, butwhich can express human immunoglobulin genes. For example, the humanheavy and light chain immunoglobulin gene complexes may be introducedrandomly or by homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring which express human antibodies. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a polypeptide of the invention. Monoclonal antibodiesdirected against the antigen can be obtained from the immunized,transgenic mice using conventional hybridoma technology. The humanimmunoglobulin transgenes harbored by the transgenic mice rearrangeduring B cell differentiation, and subsequently undergo class switchingand somatic mutation. Thus, using such a technique, it is possible toproduce therapeutically useful IgG, IgA, IgM and IgE antibodies. For anoverview of this technology for producing human antibodies, see Lonbergand Huszar, Int. Rev. Immunol. 13:65-93 (1995). For a detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies and protocols for producing such antibodies, see,e.g., PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO96/33735; European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923;5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318;5,885,793; 5,916,771; and 5,939,598, which are incorporated by referenceherein in their entirety. In addition, companies such as Abgenix, Inc.(Freemont, Calif.) and Genpharm (San Jose, Calif.) can be engaged toprovide human antibodies directed against a selected antigen usingtechnology similar to that described above.

[0150] Completely human antibodies which recognize a selected epitopecan be generated using a technique referred to as “guided selection.” Inthis approach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al., Bio/technology 12:899-903(1988)).

[0151] Further, antibodies to the polypeptides of the invention can, inturn, be utilized to generate anti-idiotype antibodies that “mimic”polypeptides of the invention using techniques well known to thoseskilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444;(1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example,antibodies which bind to and competitively inhibit polypeptidemultimerization and/or binding of a polypeptide of the invention to aligand can be used to generate anti-idiotypes that “mimic” thepolypeptide multimerization and/or binding domain and, as a consequence,bind to and neutralize polypeptide and/or its ligand. Such neutralizinganti-idiotypes or Fab fragments of such anti-idiotypes can be used intherapeutic regimens to neutralize polypeptide ligand. For example, suchanti-idiotypic antibodies can be used to bind a polypeptide of theinvention and/or to bind its ligands/receptors, and thereby block itsbiological activity.

[0152] Polynucleotides Encoding Antibodies

[0153] The invention further provides polynucleotides comprising anucleotide sequence encoding an antibody of the invention and fragmentsthereof. The invention also encompasses polynucleotides that hybridizeunder stringent or lower stringency hybridization conditions, e.g., asdefined supra, to polynucleotides that encode an antibody, preferably,that specifically binds to a polypeptide of the invention, preferably,an antibody that binds to a polypeptide having the amino acid sequenceof SEQ ID NO:2.

[0154] The polynucleotides may be obtained, and the nucleotide sequenceof the polynucleotides determined, by any method known in the art. Forexample, if the nucleotide sequence of the antibody is known, apolynucleotide encoding the antibody may be assembled from chemicallysynthesized oligonucleotides (e.g., as described in Kutmeier et al.,BioTechniques 17:242 (1994)), which, briefly, involves the synthesis ofoverlapping oligonucleotides containing portions of the sequenceencoding the antibody, annealing and ligating of those oligonucleotides,and then amplification of the ligated oligonucleotides by PCR.

[0155] Alternatively, a polynucleotide encoding an antibody may begenerated from nucleic acid from a suitable source. If a clonecontaining a nucleic acid encoding a particular antibody is notavailable, but the sequence of the antibody molecule is known, a nucleicacid encoding the immunoglobulin may be chemically synthesized orobtained from a suitable source (e.g., an antibody cDNA library, or acDNA library generated from, or nucleic acid, preferably poly A+ RNA,isolated from, any tissue or cells expressing the antibody, such ashybridoma cells selected to express an antibody of the invention) by PCRamplification using synthetic primers hybridizable to the 3′ and 5′ endsof the sequence or by cloning using an oligonucleotide probe specificfor the particular gene sequence to identify, e.g., a cDNA clone from acDNA library that encodes the antibody. Amplified nucleic acidsgenerated by PCR may then be cloned into replicable cloning vectorsusing any method well known in the art.

[0156] Once the nucleotide sequence and corresponding amino acidsequence of the antibody is determined, the nucleotide sequence of theantibody may be manipulated using methods well known in the art for themanipulation of nucleotide sequences, e.g., recombinant DNA techniques,site directed mutagenesis, PCR, etc. (see, for example, the techniquesdescribed in Sambrook et al., 1990, Molecular Cloning, A LaboratoryManual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NYand Ausubel et al., eds., 1998, Current Protocols in Molecular Biology,John Wiley & Sons, NY, which are both incorporated by reference hereinin their entireties ), to generate antibodies having a different aminoacid sequence, for example to create amino acid substitutions,deletions, and/or insertions.

[0157] In a specific embodiment, the amino acid sequence of the heavyand/or light chain variable domains may be inspected to identify thesequences of the complementarity determining regions (CDRs) by methodsthat are well know in the art, e.g., by comparison to known amino acidsequences of other heavy and light chain variable regions to determinethe regions of sequence hypervariability. Using routine recombinant DNAtechniques, one or more of the CDRs may be inserted within frameworkregions, e.g., into human framework regions to humanize a non-humanantibody, as described supra. The framework regions may be naturallyoccurring or consensus framework regions, and preferably human frameworkregions (see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479 (1998)for a listing of human framework regions). Preferably, thepolynucleotide generated by the combination of the framework regions andCDRs encodes an antibody that specifically binds a polypeptide of theinvention. Preferably, as discussed supra, one or more amino acidsubstitutions may be made within the framework regions, and, preferably,the amino acid substitutions improve binding of the antibody to itsantigen. Additionally, such methods may be used to make amino acidsubstitutions or deletions of one or more variable region cysteineresidues participating in an intrachain disulfide bond to generateantibody molecules lacking one or more intrachain disulfide bonds. Otheralterations to the polynucleotide are encompassed by the presentinvention and within the skill of the art.

[0158] In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984);Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature314:452-454 (1985)) by splicing genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Asdescribed supra, a chimeric antibody is a molecule in which differentportions are derived from different animal species, such as those havinga variable region derived from a murine mAb and a human immunoglobulinconstant region, e.g., humanized antibodies.

[0159] Alternatively, techniques described for the production of singlechain antibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423-42(1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988);and Ward et al., Nature 334:544-54 (1989)) can be adapted to producesingle chain antibodies. Single chain antibodies are formed by linkingthe heavy and light chain fragments of the Fv region via an amino acidbridge, resulting in a single chain polypeptide. Techniques for theassembly of functional Fv fragments in E. coli may also be used (Skerraet al., Science 242:1038-1041 (1988)).

[0160] Methods of Producing Antibodies

[0161] The antibodies of the invention can be produced by any methodknown in the art for the synthesis of antibodies, in particular, bychemical synthesis or preferably, by recombinant expression techniques.

[0162] Recombinant expression of an antibody of the invention, orfragment, derivative or analog thereof, (e.g., a heavy or light chain ofan antibody of the invention or a single chain antibody of theinvention), requires construction of an expression vector containing apolynucleotide that encodes the antibody. Once a polynucleotide encodingan antibody molecule or a heavy or light chain of an antibody, orportion thereof (preferably containing the heavy or light chain variabledomain), of the invention has been obtained, the vector for theproduction of the antibody molecule may be produced by recombinant DNAtechnology using techniques well known in the art. Thus, methods forpreparing a protein by expressing a polynucleotide containing anantibody encoding nucleotide sequence are described herein. Methodswhich are well known to those skilled in the art can be used toconstruct expression vectors containing antibody coding sequences andappropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. The invention,thus, provides replicable vectors comprising a nucleotide sequenceencoding an antibody molecule of the invention, or a heavy or lightchain thereof, or a heavy or light chain variable domain, operablylinked to a promoter. Such vectors may include the nucleotide sequenceencoding the constant region of the antibody molecule (see, e.g., PCTPublication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No.5,122,464) and the variable domain of the antibody may be cloned intosuch a vector for expression of the entire heavy or light chain.

[0163] The expression vector is transferred to a host cell byconventional techniques and the transfected cells are then cultured byconventional techniques to produce an antibody of the invention. Thus,the invention includes host cells containing a polynucleotide encodingan antibody of the invention, or a heavy or light chain thereof, or asingle chain antibody of the invention, operably linked to aheterologous promoter. In preferred embodiments for the expression ofdouble-chained antibodies, vectors encoding both the heavy and lightchains may be co-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below.

[0164] A variety of host-expression vector systems may be utilized toexpress the antibody molecules of the invention. Such host-expressionsystems represent vehicles by which the coding sequences of interest maybe produced and subsequently purified, but also represent cells whichmay, when transformed or transfected with the appropriate nucleotidecoding sequences, express an antibody molecule of the invention in situ.These include but are not limited to microorganisms such as bacteria(e.g., E. coli, B. subtilis) transformed with recombinant bacteriophageDNA, plasmid DNA or cosmid DNA expression vectors containing antibodycoding sequences; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing antibody codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing antibody codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing antibody coding sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells (e.g., metallothionein promoter) or from mammalianviruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5Kpromoter). Preferably, bacterial cells such as Escherichia coli, andmore preferably, eukaryotic cells, especially for the expression ofwhole recombinant antibody molecule, are used for the expression of arecombinant antibody molecule. For example, mammalian cells such asChinese hamster ovary cells (CHO), in conjunction with a vector such asthe major intermediate early gene promoter element from humancytomegalovirus is an effective expression system for antibodies(Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2(1990)).

[0165] In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791(1983)), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lac Z coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, NucleicAcids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem.24:5503-5509 (1989)); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding tomatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

[0166] In an insect system, Autographa californica nuclear polyhedrosisvirus (AcNPV) is used as a vector to express foreign genes. The virusgrows in Spodoptera frugiperda cells. The antibody coding sequence maybe cloned individually into non- essential regions (for example thepolyhedrin gene) of the virus and placed under control of an AcNPVpromoter (for example the polyhedrin promoter).

[0167] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, the antibody coding sequence of interest may beligated to an adenovirus transcription/translation control complex,e.g., the late promoter and tripartite leader sequence. This chimericgene may then be inserted in the adenovirus genome by in vitro or invivo recombination. Insertion in a non- essential region of the viralgenome (e.g., region E1 or E3) will result in a recombinant virus thatis viable and capable of expressing the antibody molecule in infectedhosts. (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA 81:355-359(1984)). Specific initiation signals may also be required for efficienttranslation of inserted antibody coding sequences. These signals includethe ATG initiation codon and adjacent sequences. Furthermore, theinitiation codon must be in phase with the reading frame of the desiredcoding sequence to ensure translation of the entire insert. Theseexogenous translational control signals and initiation codons can be ofa variety of origins, both natural and synthetic. The efficiency ofexpression may be enhanced by the inclusion of appropriate transcriptionenhancer elements, transcription terminators, etc. (see Bittner et al.,Methods in Enzymol. 153:51-544 (1987)).

[0168] In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post- translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK,293, 3T3, W138, and in particular, breast cancer cell lines such as, forexample, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary glandcell line such as, for example, CRL7030 and Hs578Bst.

[0169] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines which stablyexpress the antibody molecule may be engineered. Rather than usingexpression vectors which contain viral origins of replication, hostcells can be transformed with DNA controlled by appropriate expressioncontrol elements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the antibodymolecule. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that interact directly orindirectly with the antibody molecule.

[0170] A number of selection systems may be used, including but notlimited to the herpes simplex virus thymidine kinase (Wigler et al.,Cell 11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase(Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), andadenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980))genes can be employed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl.Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072(1981)); neo, which confers resistance to the aminoglycoside G-418Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991);Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan,Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem.62:191-217 (1993); May, 1993, TIB TECH 11(5):155-215); and hygro, whichconfers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)).Methods commonly known in the art of recombinant DNA technology may beroutinely applied to select the desired recombinant clone, and suchmethods are described, for example, in Ausubel et al. (eds.), CurrentProtocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler,Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY(1990); and in Chapters 12 and 13, Dracopoli et al. (eds), CurrentProtocols in Human Genetics, John Wiley & Sons, NY (1994);Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which areincorporated by reference herein in their entireties.

[0171] The expression levels of an antibody molecule can be increased byvector amplification (for a review, see Bebbington and Hentschel, Theuse of vectors based on gene amplification for the expression of clonedgenes in mammalian cells in DNA cloning, Vol.3. (Academic Press, NewYork, 1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., Mol. Cell. Biol. 3:257(1983)).

[0172] The host cell may be co-transfected with two expression vectorsof the invention, the first vector encoding a heavy chain derivedpolypeptide and the second vector encoding a light chain derivedpolypeptide. The two vectors may contain identical selectable markerswhich enable equal expression of heavy and light chain polypeptides.Alternatively, a single vector may be used which encodes, and is capableof expressing, both heavy and light chain polypeptides. In suchsituations, the light chain should be placed before the heavy chain toavoid an excess of toxic free heavy chain (Proudfoot, Nature 322:52(1986); Kohler, Proc. Natl. Acad. Sci. USA 77:2197 (1980)). The codingsequences for the heavy and light chains may comprise cDNA or genomicDNA.

[0173] Once an antibody molecule of the invention has been produced byan animal, chemically synthesized, or recombinantly expressed, it may bepurified by any method known in the art for purification of animmunoglobulin molecule, for example, by chromatography (e.g., ionexchange, affinity, particularly by affinity for the specific antigenafter Protein A, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins. In addition, the antibodies of the presentinvention or fragments thereof can be fused to heterologous polypeptidesequences described herein or otherwise known in the art, to facilitatepurification.

[0174] The present invention encompasses antibodies recombinantly fusedor chemically conjugated (including both covalently and non-covalentlyconjugations) to a polypeptide (or portion thereof, preferably at least10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of thepolypeptide) of the present invention to generate fusion proteins. Thefusion does not necessarily need to be direct, but may occur throughlinker sequences. The antibodies may be specific for antigens other thanpolypeptides (or portion thereof, preferably at least 10, 20, 30, 40,50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the presentinvention. For example, antibodies may be used to target thepolypeptides of the present invention to particular cell types, eitherin vitro or in vivo, by fusing or conjugating the polypeptides of thepresent invention to antibodies specific for particular cell surfacereceptors. Antibodies fused or conjugated to the polypeptides of thepresent invention may also be used in in vitro immunoassays andpurification methods using methods known in the art. See e.g., Harbor etal., supra, and PCT publication WO 93/21232; EP 439,095; Naramura etal., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No. 5,474,981; Gillies etal., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol.146:2446-2452(1991), which are incorporated by reference in theirentireties.

[0175] The present invention further includes compositions comprisingthe polypeptides of the present invention fused or conjugated toantibody domains other than the variable regions. For example, thepolypeptides of the present invention may be fused or conjugated to anantibody Fc region, or portion thereof. The antibody portion fused to apolypeptide of the present invention may comprise the constant region,hinge region, CH1 domain, CH2 domain, and CH3 domain or any combinationof whole domains or portions thereof. The polypeptides may also be fusedor conjugated to the above antibody portions to form multimers. Forexample, Fc portions fused to the polypeptides of the present inventioncan form dimers through disulfide bonding between the Fc portions.Higher multimeric forms can be made by fusing the polypeptides toportions of IgA and IgM. Methods for fusing or conjugating thepolypeptides of the present invention to antibody portions are known inthe art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046;5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166; PCTpublications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc. Natl.Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J. Immunol.154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA89:11337-11341(1992) (said references incorporated by reference in theirentireties).

[0176] As discussed, supra, the polypeptides corresponding to apolypeptide, polypeptide fragment, or a variant of SEQ ID NO:2 may befused or conjugated to the above antibody portions to increase the invivo half life of the polypeptides or for use in immunoassays usingmethods known in the art. Further, the polypeptides corresponding to SEQID NO:2 may be fused or conjugated to the above antibody portions tofacilitate purification. One reported example describes chimericproteins consisting of the first two domains of the humanCD4-polypeptide and various domains of the constant regions of the heavyor light chains of mammalian immunoglobulins. (EP 394,827; Traunecker etal., Nature 331:84-86 (1988). The polypeptides of the present inventionfused or conjugated to an antibody having disulfide- linked dimericstructures (due to the IgG) may also be more efficient in binding andneutralizing other molecules, than the monomeric secreted protein orprotein fragment alone. (Fountoulakis et al., J. Biochem. 270:3958-3964(1995)). In many cases, the Fc part in a fusion protein is beneficial intherapy and diagnosis, and thus can result in, for example, improvedpharmacokinetic properties. (EP A 232,262). Alternatively, deleting theFc part after the fusion protein has been expressed, detected, andpurified, would be desired. For example, the Fc portion may hindertherapy and diagnosis if the fusion protein is used as an antigen forimmunizations. In drug discovery, for example, human proteins, such ashIL-5, have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. (See,Bennett et al., J. Molecular Recognition 8:52-58 (1995); Johanson etal., J. Biol. Chem. 270:9459-9471 (1995).

[0177] Moreover, the antibodies or fragments thereof of the presentinvention can be fused to marker sequences, such as a peptide tofacilitate purification. In preferred embodiments, the marker amino acidsequence is a hexa-histidine peptide, such as the tag provided in a pQEvector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311),among others, many of which are commercially available. As described inGentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), forinstance, hexa- histidine provides for convenient purification of thefusion protein. Other peptide tags useful for purification include, butare not limited to, the “HA” tag, which corresponds to an epitopederived from the influenza hemagglutinin protein (Wilson et al., Cell37:767 (1984)) and the “flag” tag.

[0178] The present invention further encompasses antibodies or fragmentsthereof conjugated to a diagnostic or therapeutic agent. The antibodiescan be used diagnostically to, for example, monitor the development orprogression of a tumor as part of a clinical testing procedure to, e.g.,determine the efficacy of a given treatment regimen. Detection can befacilitated by coupling the antibody to a detectable substance. Examplesof detectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,radioactive materials, positron emitting metals using various positronemission tomographies, and nonradioactive paramagnetic metal ions. Thedetectable substance may be coupled or conjugated either directly to theantibody (or fragment thereof) or indirectly, through an intermediate(such as, for example, a linker known in the art) using techniques knownin the art. See, for example, U.S. Pat. No. 4,741,900 for metal ionswhich can be conjugated to antibodies for use as diagnostics accordingto the present invention. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidinibiotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude 125I, 131I, 111In or 99Tc.

[0179] Further, an antibody or fragment thereof may be conjugated to atherapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidalagent, a therapeutic agent or a radioactive metal ion, e.g.,alpha-emitters such as, for example, 213Bi. A cytotoxin or cytotoxicagent includes any agent that is detrimental to cells. Examples includepaclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

[0180] The conjugates of the invention can be used for modifying a givenbiological response, the therapeutic agent or drug moiety is not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, a-interferon, β-interferon,nerve growth factor, platelet derived growth factor, tissue plasminogenactivator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I (See,International Publication No. WO 97/33899), AIM II (See, InternationalPublication No. WO 97/34911), Fas Ligand (Takahashi et al., Int.Immunol., 6:1567-1574 (1994)), VEGI (See, International Publication No.WO 99/23105), a thrombotic agent or an anti- angiogenic agent, e.g.,angiostatin or endostatin; or, biological response modifiers such as,for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

[0181] Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

[0182] Techniques for conjugating such therapeutic moiety to antibodiesare well known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev. 62:119-58 (1982).

[0183] Alternatively, an antibody can be conjugated to a second antibodyto form an antibody heteroconjugate as described by Segal in U.S. Pat.No. 4,676,980, which is incorporated herein by reference in itsentirety.

[0184] An antibody, with or without a therapeutic moiety conjugated toit, administered alone or in combination with cytotoxic factor(s) and/orcytokine(s) can be used as a therapeutic.

[0185] Immunophenotyping

[0186] The antibodies of the invention may be utilized forimmunophenotyping of cell lines and biological samples. The translationproduct of the gene of the present invention may be useful as a cellspecific marker, or more specifically as a cellular marker that isdifferentially expressed at various stages of differentiation and/ormaturation of particular cell types. Monoclonal antibodies directedagainst a specific epitope, or combination of epitopes, will allow forthe screening of cellular populations expressing the marker. Varioustechniques can be utilized using monoclonal antibodies to screen forcellular populations expressing the marker(s), and include magneticseparation using antibody-coated magnetic beads, “panning” with antibodyattached to a solid matrix (i.e., plate), and flow cytometry (See, e.g.,U.S. Pat. No. 5,985,660; and Morrison et al., Cell, 96:737-49 (1999)).

[0187] These techniques allow for the screening of particularpopulations of cells, such as might be found with hematologicalmalignancies (i.e. minimal residual disease (MRD) in acute leukemicpatients) and “non-self” cells in transplantations to preventGraft-versus-Host Disease (GVHD). Alternatively, these techniques allowfor the screening of hematopoietic stem and progenitor cells capable ofundergoing proliferation and/or differentiation, as might be found inhuman umbilical cord blood.

[0188] Assays For Antibody Binding

[0189] The antibodies of the invention may be assayed for immunospecificbinding by any method known in the art. The immunoassays which can beused include but are not limited to competitive and non-competitiveassay systems using techniques such as western blots, radioimmunoassays,ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, to name but a few. Such assays areroutine and well known in the art (see, e.g., Ausubel et al, eds, 1994,Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc.,New York, which is incorporated by reference herein in its entirety).Exemplary immunoassays are described briefly below (but are not intendedby way of limitation).

[0190] Immunoprecipitation protocols generally comprise lysing apopulation of cells in a lysis buffer such as RIPA buffer (1% NP-40 orTriton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 Msodium phosphate at pH 7.2, 1% Trasylol) supplemented with proteinphosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin,sodium vanadate), adding the antibody of interest to the cell lysate,incubating for a period of time (e.g., 1-4 hours) at 4° C., addingprotein A and/or protein G sepharose beads to the cell lysate,incubating for about an hour or more at 4° C, washing the beads in lysisbuffer and resuspending the beads in SDS/sample buffer. The ability ofthe antibody of interest to immunoprecipitate a particular antigen canbe assessed by, e.g., western blot analysis. One of skill in the artwould be knowledgeable as to the parameters that can be modified toincrease the binding of the antibody to an antigen and decrease thebackground (e.g., pre-clearing the cell lysate with sepharose beads).For further discussion regarding immunoprecipitation protocols see,e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology,Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.

[0191] Western blot analysis generally comprises preparing proteinsamples, electrophoresis of the protein samples in a polyacrylamide gel(e.g., 8%-20% SDS-PAGE depending on the molecular weight of theantigen), transferring the protein sample from the polyacrylamide gel toa membrane such as nitrocellulose, PVDF or nylon, blocking the membranein blocking solution (e.g., PBS with 3% BSA or non-fat milk), washingthe membrane in washing buffer (e.g., PBS-Tween 20), blocking themembrane with primary antibody (the antibody of interest) diluted inblocking buffer, washing the membrane in washing buffer, blocking themembrane with a secondary antibody (which recognizes the primaryantibody, e.g., an anti-human antibody) conjugated to an enzymaticsubstrate (e.g., horseradish peroxidase or alkaline phosphatase) orradioactive molecule (e.g., 32P or 1251) diluted in blocking buffer,washing the membrane in wash buffer, and detecting the presence of theantigen. One of skill in the art would be knowledgeable as to theparameters that can be modified to increase the signal detected and toreduce the background noise. For further discussion regarding westernblot protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols inMolecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.8.1.

[0192] ELISAs comprise preparing antigen, coating the well of a 96 wellmicrotiter plate with the antigen, adding the antibody of interestconjugated to a detectable compound such as an enzymatic substrate(e.g., horseradish peroxidase or alkaline phosphatase) to the well andincubating for a period of time, and detecting the presence of theantigen. In ELISAs the antibody of interest does not have to beconjugated to a detectable compound; instead, a second antibody (whichrecognizes the antibody of interest) conjugated to a detectable compoundmay be added to the well. Further, instead of coating the well with theantigen, the antibody may be coated to the well. In this case, a secondantibody conjugated to a detectable compound may be added following theaddition of the antigen of interest to the coated well. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected as well as other variations of ELISAsknown in the art. For further discussion regarding ELISAs see, e.g.,Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol.1, John Wiley & Sons, Inc., New York at 11.2.1.

[0193] The binding affinity of an antibody to an antigen and theoff-rate of an antibody-antigen interaction can be determined bycompetitive binding assays. One example of a competitive binding assayis a radioimmunoassay comprising the incubation of labeled antigen(e.g., 3H or 1251) with the antibody of interest in the presence ofincreasing amounts of unlabeled antigen, and the detection of theantibody bound to the labeled antigen. The affinity of the antibody ofinterest for a particular antigen and the binding off-rates can bedetermined from the data by scatchard plot analysis. Competition with asecond antibody can also be determined using radioimmunoassays. In thiscase, the antigen is incubated with antibody of interest conjugated to alabeled compound (e.g., 3H or 125I) in the presence of increasingamounts of an unlabeled second antibody.

[0194] Therapeutic Uses

[0195] The present invention is further directed to antibody-basedtherapies which involve administering antibodies of the invention to ananimal, preferably a mammal, and most preferably a human, patient fortreating one or more of the disclosed diseases, disorders, orconditions. Therapeutic compounds of the invention include, but are notlimited to, antibodies of the invention (including fragments, analogsand derivatives thereof as described herein) and nucleic acids encodingantibodies of the invention (including fragments, analogs andderivatives thereof and anti-idiotypic antibodies as described herein).The antibodies of the invention can be used to treat, inhibit or preventdiseases, disorders or conditions associated with aberrant expressionand/or activity of a polypeptide of the invention, including, but notlimited to, any one or more of the diseases, disorders, or conditionsdescribed herein. The treatment and/or prevention of diseases,disorders, or conditions associated with aberrant expression and/oractivity of a polypeptide of the invention includes, but is not limitedto, alleviating symptoms associated with those diseases, disorders orconditions. Antibodies of the invention may be provided inpharmaceutically acceptable compositions as known in the art or asdescribed herein.

[0196] A summary of the ways in which the antibodies of the presentinvention may be used therapeutically includes binding polynucleotidesor polypeptides of the present invention locally or systemically in thebody or by direct cytotoxicity of the antibody, e.g. as mediated bycomplement (CDC) or by effector cells (ADCC). Some of these approachesare described in more detail below. Armed with the teachings providedherein, one of ordinary skill in the art will know how to use theantibodies of the present invention for diagnostic, monitoring ortherapeutic purposes without undue experimentation.

[0197] The antibodies of this invention may be advantageously utilizedin combination with other monoclonal or chimeric antibodies, or withlymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3and IL-7), for example, which serve to increase the number or activityof effector cells which interact with the antibodies.

[0198] The antibodies of the invention may be administered alone or incombination with other types of treatments (e.g., radiation therapy,chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents).Generally, administration of products of a species origin or speciesreactivity (in the case of antibodies) that is the same species as thatof the patient is preferred. Thus, in a preferred embodiment, humanantibodies, fragments derivatives, analogs, or nucleic acids, areadministered to a human patient for therapy or prophylaxis.

[0199] It is preferred to use high affinity and/or potent in vivoinhibiting and/or neutralizing antibodies against polypeptides orpolynucleotides of the present invention, fragments or regions thereof,for both immunoassays directed to and therapy of disorders related topolynucleotides or polypeptides, including fragments thereof, of thepresent invention. Such antibodies, fragments, or regions, willpreferably have an affinity for polynucleotides or polypeptides of theinvention, including fragments thereof. Preferred binding affinitiesinclude those with a dissociation constant or Kd less than 5×10⁻² M,10⁻² M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M,10⁻⁶ M, 5×10⁻⁷ M, 10 ⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M,5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, and 10⁻¹⁵ M.

[0200] Gene Therapy

[0201] In a specific embodiment, nucleic acids comprising sequencesencoding antibodies or functional derivatives thereof, are administeredto treat, inhibit or prevent a disease or disorder associated withaberrant expression and/or activity of a polypeptide of the invention,by way of gene therapy. Additionally, nucleic acids comprising sequencesencoding BAIT polypeptides of the invention, are administered to treat,inhibit, or prevent a disease or disorder associated with aberrantexpression and/or activity of a polypeptide of the invention, by way ofgene therapy.

[0202] Gene therapy refers to therapy performed by the administration toa subject of an expressed or expressible nucleic acid. In thisembodiment of the invention, the nucleic acids produce their encodedprotein that mediates a therapeutic effect.

[0203] Any of the methods for gene therapy available in the art can beused according to the present invention. Exemplary methods are describedbelow.

[0204] For general reviews of the methods of gene therapy, see Goldspielet al., Clinical Pharmacy 12:488-505 (1993); Wu and Wu, Biotherapy3:87-95 (1991);

[0205] Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993);Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev.Biochem. 62:191-217 (1993); May, TIBTECH 11(5):155-215 (1993). Methodscommonly known in the art of recombinant DNA technology which can beused are described in Ausubel et al. (eds.), Current Protocols inMolecular Biology, John Wiley & Sons, NY (1993); and Kriegler, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

[0206] In a preferred aspect, the compound comprises nucleic acidsequences encoding an antibody, said nucleic acid sequences being partof expression vectors that express the antibody or fragments or chimericproteins or heavy or light chains thereof in a suitable host. Inparticular, such nucleic acid sequences have promoters operably linkedto the antibody coding region, said promoter being inducible orconstitutive, and, optionally, tissue- specific. In another particularembodiment, nucleic acid molecules are used in which the antibody codingsequences and any other desired sequences are flanked by regions thatpromote homologous recombination at a desired site in the genome, thusproviding for intrachromosomal expression of the antibody encodingnucleic acids (Koller and Smithies, Proc. Natl. Acad. Sci. USA86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989). Inspecific embodiments, the expressed antibody molecule is a single chainantibody; alternatively, the nucleic acid sequences include sequencesencoding both the heavy and light chains, or fragments thereof, of theantibody.

[0207] Delivery of the nucleic acids into a patient may be eitherdirect, in which case the patient is directly exposed to the nucleicacid or nucleic acid- carrying vectors, or indirect, in which case,cells are first transformed with the nucleic acids in vitro, thentransplanted into the patient. These two approaches are known,respectively, as in vivo or ex vivo gene therapy.

[0208] In a specific embodiment, the nucleic acid sequences are directlyadministered in vivo, where it is expressed to produce the encodedproduct. This can be accomplished by any of numerous methods known inthe art, e.g., by constructing them as part of an appropriate nucleicacid expression vector and administering it so that they becomeintracellular, e.g., by infection using defective or attenuatedretrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or bydirect injection of naked DNA, or by use of microparticle bombardment(e.g., a gene gun; Biolistic, Dupont), or coating with lipids orcell-surface receptors or transfecting agents, encapsulation inliposomes, microparticles, or microcapsules, or by administering them inlinkage to a peptide which is known to enter the nucleus, byadministering it in linkage to a ligand subject to receptor-mediatedendocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987))(which can be used to target cell types specifically expressing thereceptors), etc. In another embodiment, nucleic acid-ligand complexescan be formed in which the ligand comprises a fusogenic viral peptide todisrupt endosomes, allowing the nucleic acid to avoid lysosomaldegradation. In yet another embodiment, the nucleic acid can be targetedin vivo for cell specific uptake and expression, by targeting a specificreceptor (see, e.g., PCT Publications WO 92/06180; WO 92/22635;WO92/20316; WO93/14188, WO 93/20221). Alternatively, the nucleic acidcan be introduced intracellularly and incorporated within host cell DNAfor expression, by homologous recombination (Koller and Smithies, Proc.Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature342:435-438 (1989)).

[0209] In a specific embodiment, viral vectors that contains nucleicacid sequences encoding an antibody of the invention are used. Forexample, a retroviral vector can be used (see Miller et al., Meth.Enzymol. 217:581-599 (1993)). These retroviral vectors contain thecomponents necessary for the correct packaging of the viral genome andintegration into the host cell DNA. The nucleic acid sequences encodingthe antibody to be used in gene therapy are cloned into one or morevectors, which facilitates delivery of the gene into a patient. Moredetail about retroviral vectors can be found in Boesen et al.,Biotherapy 6:291-302 (1994), which describes the use of a retroviralvector to deliver the mdr1 gene to hematopoietic stem cells in order tomake the stem cells more resistant to chemotherapy. Other referencesillustrating the use of retroviral vectors in gene therapy are: Cloweset al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141(1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel.3:110-114 (1993).

[0210] Adenoviruses are other viral vectors that can be used in genetherapy. Adenoviruses are especially attractive vehicles for deliveringgenes to respiratory epithelia. Adenoviruses naturally infectrespiratory epithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, CurrentOpinion in Genetics and Development 3:499-503 (1993) present a review ofadenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10(1994) demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al.,Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992);

[0211] Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCTPublication WO94/12649;

[0212] and Wang, et al., Gene Therapy 2:775-783 (1995). In a preferredembodiment, adenovirus vectors are used.

[0213] Adeno-associated virus (AAV) has also been proposed for use ingene therapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300(1993); U.S. Pat. No. 5,436,146).

[0214] Another approach to gene therapy involves transferring a gene tocells in tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient.

[0215] In this embodiment, the nucleic acid is introduced into a cellprior to administration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol.217:599-618 (1993); Cohen et al., Meth. Enzymol. 217:618-644 (1993);Cline, Pharmac. Ther. 29:69-92m (1985) and may be used in accordancewith the present invention, provided that the necessary developmentaland physiological functions of the recipient cells are not disrupted.The technique should provide for the stable transfer of the nucleic acidto the cell, so that the nucleic acid is expressible by the cell andpreferably heritable and expressible by its cell progeny.

[0216] The resulting recombinant cells can be delivered to a patient byvarious methods known in the art. Recombinant blood cells (e.g.,hematopoietic stem or progenitor cells) are preferably administeredintravenously. The amount of cells envisioned for use depends on thedesired effect, patient state, etc., and can be determined by oneskilled in the art.

[0217] Cells into which a nucleic acid can be introduced for purposes ofgene therapy encompass any desired, available cell type, and include butare not limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such asTlymphocytes, Blymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

[0218] In a preferred embodiment, the cell used for gene therapy isautologous to the patient.

[0219] In an embodiment in which recombinant cells are used in genetherapy, nucleic acid sequences encoding an antibody are introduced intothe cells such that they are expressible by the cells or their progeny,and the recombinant cells are then administered in vivo for therapeuticeffect. In a specific embodiment, stem or progenitor cells are used. Anystem andlor progenitor cells which can be isolated and maintained invitro can potentially be used in accordance with this embodiment of thepresent invention (see e.g. PCT Publication WO 94/08598; Stemple andAnderson, Cell 71:973-985 (1992); Rheinwald, Meth. Cell Bio. 21A: 229(1980); and Pittelkow and Scott, Mayo Clinic Proc. 61:771 (1986)).

[0220] In a specific embodiment, the nucleic acid to be introduced forpurposes of gene therapy comprises an inducible promoter operably linkedto the coding region, such that expression of the nucleic acid iscontrollable by controlling the presence or absence of the appropriateinducer of transcription.

[0221] Demonstration of Therapeutic or Prophylactic Activity

[0222] The compounds or pharmaceutical compositions of the invention arepreferably tested in vitro, and then in vivo for the desired therapeuticor prophylactic activity, prior to use in humans. For example, in vitroassays to demonstrate the therapeutic or prophylactic utility of acompound or pharmaceutical composition include, the effect of a compoundon a cell line or a patient tissue sample. The effect of the compound orcomposition on the cell line and/or tissue sample can be determinedutilizing techniques known to those of skill in the art including, butnot limited to, rosette formation assays and cell lysis assays. Inaccordance with the invention, in vitro assays which can be used todetermine whether administration of a specific compound is indicated,include in vitro cell culture assays in which a patient tissue sample isgrown in culture, and exposed to or otherwise administered a compound,and the effect of such compound upon the tissue sample is observed.

[0223] Therapeutic/Prophylactic Administration and Composition

[0224] The invention provides methods of treatment, inhibition andprophylaxis by administration to a subject of an effective amount of acompound or pharmaceutical composition of the invention, preferably anantibody of the invention. In a preferred aspect, the compound issubstantially purified (e.g., substantially free from substances thatlimit its effect or produce undesired side-effects). The subject ispreferably an animal, including but not limited to animals such as cows,pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal,and most preferably human.

[0225] Formulations and methods of administration that can be employedwhen the compound comprises a nucleic acid or an immunoglobulin aredescribed above; additional appropriate formulations and routes ofadministration can be selected from among those described herein below.

[0226] Various delivery systems are known and can be used to administera compound of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J.Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid aspart of a retroviral or other vector, etc. Methods of introductioninclude but are not limited to intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The compounds or compositions may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local. In addition, it may be desirable to introduce thepharmaceutical compounds or compositions of the invention into thecentral nervous system by any suitable route, including intraventricularand intrathecal injection; intraventricular injection may be facilitatedby an intraventricular catheter, for example, attached to a reservoir,such as an Ommaya reservoir. Pulmonary administration can also beemployed, e.g., by use of an inhaler or nebulizer, and formulation withan aerosolizing agent.

[0227] In a specific embodiment, it may be desirable to administer thepharmaceutical compounds or compositions of the invention locally to thearea in need of treatment; this may be achieved by, for example, and notby way of limitation, local infusion during surgery, topicalapplication, e.g., in conjunction with a wound dressing after surgery,by injection, by means of a catheter, by means of a suppository, or bymeans of an implant, said implant being of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers. Preferably, when administering a protein, including anantibody, of the invention, care must be taken to use materials to whichthe protein does not absorb.

[0228] In another embodiment, the compound or composition can bedelivered in a vesicle, in particular a liposome (see Langer, Science249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy ofInfectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss,N.Y., pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; seegenerally ibid.)

[0229] In yet another embodiment, the compound or composition can bedelivered in a controlled release system. In one embodiment, a pump maybe used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201(1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl.J. Med. 321:574 (1989)). In another embodiment, polymeric materials canbe used (see Medical Applications of Controlled Release, Langer and Wise(eds.), CRC Pres., Boca Raton, Florida (1974); Controlled DrugBioavailability, Drug Product Design and Performance, Smolen and Ball(eds.), Wiley, New York (1984); Ranger and Peppas, J., Macromol. Sci.Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190(1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al.,J.Neurosurg. 71:105 (1989)). In yet another embodiment, a controlledrelease system can be placed in proximity of the therapeutic target,i.e., the brain, thus requiring only a fraction of the systemic dose(see, e.g., Goodson, in Medical Applications of Controlled Release,supra, vol. 2, pp. 115-138 (1984)).

[0230] Other controlled release systems are discussed in the review byLanger (Science 249:1527-1533 (1990)).

[0231] In a specific embodiment where the compound of the invention is anucleic acid encoding a protein, the nucleic acid can be administered invivo to promote expression of its encoded protein, by constructing it aspart of an appropriate nucleic acid expression vector and administeringit so that it becomes intracellular, e.g., by use of a retroviral vector(see U.S. Pat. No. 4,980,286), or by direct injection, or by use ofmicroparticle bombardment (e.g., a gene gun; Biolistic, Dupont), orcoating with lipids or cell-surface receptors or transfecting agents, orby administering it in linkage to a homeobox-like peptide which is knownto enter the nucleus (see e.g., Joliot et al., Proc. Natl. Acad. Sci.USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic acid can beintroduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination.

[0232] The present invention also provides pharmaceutical compositions.Such compositions comprise a therapeutically effective amount of acompound, and a pharmaceutically acceptable carrier. In a specificembodiment, the term “pharmaceutically acceptable” means approved by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. The term “carrier” refers to adiluent, adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions will containa therapeutically effective amount of the compound, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the patient. Theformulation should suit the mode of administration.

[0233] In a preferred embodiment, the composition is formulated inaccordance with routine procedures as a pharmaceutical compositionadapted for intravenous administration to human beings. Typically,compositions for intravenous administration are solutions in sterileisotonic aqueous buffer. Where necessary, the composition may alsoinclude a solubilizing agent and a local anesthetic such as lignocaineto ease pain at the site of the injection. Generally, the ingredientsare supplied either separately or mixed together in unit dosage form,for example, as a dry lyophilized powder or water free concentrate in ahermetically sealed container such as an ampoule or sachette indicatingthe quantity of active agent. Where the composition is to beadministered by infusion, it can be dispensed with an infusion bottlecontaining sterile pharmaceutical grade water or saline. Where thecomposition is administered by injection, an ampoule of sterile waterfor injection or saline can be provided so that the ingredients may bemixed prior to administration.

[0234] The compounds of the invention can be formulated as neutral orsalt forms. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

[0235] The amount of the compound of the invention which will beeffective in the treatment, inhibition and prevention of a disease ordisorder associated with aberrant expression and/or activity of apolypeptide of the invention can be determined by standard clinicaltechniques. In addition, in vitro assays may optionally be employed tohelp identify optimal dosage ranges. The precise dose to be employed inthe formulation will also depend on the route of administration, and theseriousness of the disease or disorder, and should be decided accordingto the judgment of the practitioner and each patient's circumstances.Effective doses may be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems.

[0236] For antibodies, the dosage administered to a patient is typically0.1 mg/kg to 100 mg/kg of the patient's body weight. Preferably, thedosage administered to a patient is between 0.1 mg/kg and 20 mg/kg ofthe patient's body weight, more preferably 1 mg/kg to 10 mg/kg of thepatient's body weight. Generally, human antibodies have a longerhalf-life within the human body than antibodies from other species dueto the immune response to the foreign polypeptides. Thus, lower dosagesof human antibodies and less frequent administration is often possible.Further, the dosage and frequency of administration of antibodies of theinvention may be reduced by enhancing uptake and tissue penetration(e.g., into the brain) of the antibodies by modifications such as, forexample, lipidation.

[0237] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention.Optionally associated with such container(s) can be a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration.

[0238] Diagnosis and Imaging

[0239] Labeled antibodies, and derivatives and analogs thereof, whichspecifically bind to a polypeptide of interest can be used fordiagnostic purposes to detect, diagnose, or monitor diseases and/ordisorders associated with the aberrant expression and/or activity of apolypeptide of the invention. The invention provides for the detectionof aberrant expression of a polypeptide of interest, comprising (a)assaying the expression of the polypeptide of interest in cells or bodyfluid of an individual using one or more antibodies specific to thepolypeptide interest and (b) comparing the level of gene expression witha standard gene expression level, whereby an increase or decrease in theassayed polypeptide gene expression level compared t the standardexpression level is indicative of aberrant expression.

[0240] The invention provides a diagnostic assay for diagnosing adisorder, comprising (a) assaying the expression of the polypeptide ofinterest in cells or body fluid of an individual using one or moreantibodies specific to the polypeptide interest and (b) comparing thelevel of gene expression with a standard gene expression level, wherebyan increase or decrease in the assayed polypeptide gene expression levelcompared to the standard expression level is indicative of a particulardisorder. With respect to cancer, the presence of a relatively highamount of transcript in biopsied tissue from an individual may indicatea predisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

[0241] Antibodies of the invention can be used to assay protein levelsin a biological sample using classical immunohistological methods knownto those of skill in the art (e.g., see Jalkanen, et al., J. Cell. Biol.101:976-985 (1985); Jalkanen, et al., J. Cell . Biol. 105:3087-3096(1987)). Other antibody-based methods useful for detecting protein geneexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assaylabels are known in the art and include enzyme labels, such as, glucoseoxidase; radioisotopes, such as iodine (125I, 121I), carbon (14C),sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc);luminescent labels, such as luminol; and fluorescent labels, such asfluorescein and rhodamine, and biotin.

[0242] One aspect of the invention is the detection and diagnosis of adisease or disorder associated with aberrant expression of a polypeptideof interest in an animal, preferably a mammal and most preferably ahuman. In one embodiment, diagnosis comprises: a) administering (forexample, parenterally, subcutaneously, or intraperitoneally) to asubject an effective amount of a labeled molecule which specificallybinds to the polypeptide of interest; b) waiting for a time intervalfollowing the administering for permitting the labeled molecule topreferentially concentrate at sites in the subject where the polypeptideis expressed (and for unbound labeled molecule to be cleared tobackground level); c) determining background level; and d) detecting thelabeled molecule in the subject, such that detection of labeled moleculeabove the background level indicates that the subject has a particulardisease or disorder associated with aberrant expression of thepolypeptide of interest. Background level can be determined by variousmethods including, comparing the amount of labeled molecule detected toa standard value previously determined for a particular system.

[0243] It will be understood in the art that the size of the subject andthe imaging system used will determine the quantity of imaging moietyneeded to produce diagnostic images. In the case of a radioisotopemoiety, for a human subject, the quantity of radioactivity injected willnormally range from about 5 to 20 millicuries of 99mTc. The labeledantibody or antibody fragment will then preferentially accumulate at thelocation of cells which contain the specific protein. In vivo tumorimaging is described in S. W. Burchiel et al., “Immunopharmacokineticsof Radiolabeled io Antibodies and Their Fragments.” Chapter 13 in TumorImaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A.Rhodes, eds., Masson Publishing Inc. (1982).

[0244] Depending on several variables, including the type of label usedand the mode of administration, the time interval following theadministration for permitting the labeled molecule to preferentiallyconcentrate at sites in the subject and for unbound labeled molecule tobe cleared to background level is 6 to 48 hours or 6 to 24 hours or 6 to12 hours. In another embodiment the time interval followingadministration is 5 to 20 days or 5 to 10 days.

[0245] In an embodiment, monitoring of the disease or disorder iscarried out by repeating the method for diagnosing the disease ordisease, for example, one month after initial diagnosis, six monthsafter initial diagnosis, one year after initial diagnosis, etc.

[0246] Presence of the labeled molecule can be detected in the patientusing methods known in the art for in vivo scanning. These methodsdepend upon the type of label used. Skilled artisans will be able todetermine the appropriate method for detecting a particular label.Methods and devices that may be used in the diagnostic methods of theinvention include, but are not limited to, computed tomography (CT),whole body scan such as position emission tomography (PET), magneticresonance imaging (MRI), and sonography.

[0247] In a specific embodiment, the molecule is labeled with aradioisotope and is detected in the patient using a radiation responsivesurgical instrument (Thurston et al., U.S. Pat. No. 5,441,050). Inanother embodiment, the molecule is labeled with a fluorescent compoundand is detected in the patient using a fluorescence responsive scanninginstrument. In another embodiment, the molecule is labeled with apositron emitting metal and is detected in the patent using positronemission-tomography. In yet another embodiment, the molecule is labeledwith a paramagnetic label and is detected in a patient using magneticresonance imaging (MRI).

[0248] Kits

[0249] The present invention provides kits that can be used in the abovemethods. In one embodiment, a kit comprises an antibody of theinvention, preferably a purified antibody, in one or more containers. Ina specific embodiment, the kits of the present invention contain asubstantially isolated polypeptide comprising an epitope which isspecifically immunoreactive with an antibody included in the kit.Preferably, the kits of the present invention further comprise a controlantibody which does not react with the polypeptide of interest. Inanother specific embodiment, the kits of the present invention contain ameans for detecting the binding of an antibody to a polypeptide ofinterest (e.g., the antibody may be conjugated to a detectable substratesuch as a fluorescent compound, an enzymatic substrate, a radioactivecompound or a luminescent compound, or a second antibody whichrecognizes the first antibody may be conjugated to a detectablesubstrate).

[0250] In another specific embodiment of the present invention, the kitis a diagnostic kit for use in screening serum containing antibodiesspecific against proliferative and/or cancerous polynucleotides andpolypeptides. Such a kit may include a control antibody that does notreact with the polypeptide of interest. Such a kit may include asubstantially isolated polypeptide antigen comprising an epitope whichis specifically immunoreactive with at least one anti-polypeptideantigen antibody. Further, such a kit includes means for detecting thebinding of said antibody to the antigen (e.g., the antibody may beconjugated to a fluorescent compound such as fluorescein or rhodaminewhich can be detected by flow cytometry). In specific embodiments, thekit may include a recombinantly produced or chemically synthesizedpolypeptide antigen. The polypeptide antigen of the kit may also beattached to a solid support.

[0251] In a more specific embodiment the detecting means of theabove-described kit includes a solid support to which said polypeptideantigen is attached. Such a kit may also include a non-attachedreporter-labeled anti-human antibody. In this embodiment, binding of theantibody to the polypeptide antigen can be detected by binding of thesaid reporter-labeled antibody.

[0252] In an additional embodiment, the invention includes a diagnostickit for use in screening serum containing antigens of the polypeptide ofthe invention. The diagnostic kit includes a substantially isolatedantibody specifically immunoreactive with polypeptide or polynucleotideantigens, and means for detecting the binding of the polynucleotide orpolypeptide antigen to the antibody. In one embodiment, the antibody isattached to a solid support. In a specific embodiment, the antibody maybe a monoclonal antibody. The detecting means of the kit may include asecond, labeled monoclonal antibody. Alternatively, or in addition, thedetecting means may include a labeled, competing antigen.

[0253] In one diagnostic configuration, test serum is reacted with asolid phase reagent having a surface-bound antigen obtained by themethods of the present invention. After binding with specific antigenantibody to the reagent and removing unbound serum components bywashing, the reagent is reacted with reporter-labeled anti-humanantibody to bind reporter to the reagent in proportion to the amount ofbound anti- antigen antibody on the solid support. The reagent is againwashed to remove unbound labeled antibody, and the amount of reporterassociated with the reagent is determined. Typically, the reporter is anenzyme which is detected by incubating the solid phase in the presenceof a suitable fluorometric, luminescent or colorimetric substrate(Sigma, St. Louis, Mo.).

[0254] The solid surface reagent in the above assay is prepared by knowntechniques for attaching protein material to solid support material,such as polymeric beads, dip sticks, 96-well plate or filter material.These attachment methods generally include non-specific adsorption ofthe protein to the support or covalent attachment of the protein,typically through a free amine group, to a chemically reactive group onthe solid support, such as an activated carboxyl, hydroxyl, or aldehydegroup. Alternatively, streptavidin coated plates can be used inconjunction with biotinylated antigen(s).

[0255] Thus, the invention provides an assay system or kit for carryingout this diagnostic method. The kit generally includes a support withsurface- bound recombinant antigens, and a reporter-labeled anti-humanantibody for detecting surface-bound anti-antigen antibody.

[0256] Fusion Proteins

[0257] Any polypeptide of the present invention can be used to generatefusion proteins. For example, the polypeptide of the present invention,when fused to a second protein, can be used as an antigenic tag.Antibodies raised against the polypeptide of the present invention canbe used to indirectly detect the second protein by binding to thepolypeptide. Moreover, because secreted proteins target cellularlocations based on trafficking signals, the polypeptides of the presentinvention can be used as targeting molecules once fused to otherproteins.

[0258] Examples of domains that can be fused to polypeptides of thepresent invention include not only heterologous signal sequences, butalso other heterologous functional regions. The fusion does notnecessarily need to be direct, but may occur through linker sequences.

[0259] Moreover, fusion proteins may also be engineered to improvecharacteristics of the polypeptide of the present invention. Forinstance, a region of additional amino acids, particularly charged aminoacids, may be added to the N-terminus of the polypeptide to improvestability and persistence during purification from the host cell orsubsequent handling and storage. Also, peptide moieties may be added tothe polypeptide to facilitate purification. Such regions may be removedprior to final preparation of the polypeptide. The addition of peptidemoieties to facilitate handling of polypeptides are familiar and routinetechniques in the art.

[0260] Moreover, polypeptides of the present invention, includingfragments, and specifically epitopes, can be combined with parts of theconstant domain of immunoglobulins (IgA, IgE, IgG, IgM) or portionsthereof (CH1, CH2, CH3, and any combination thereof, including bothentire domains and portions thereof), resulting in chimericpolypeptides. These fusion proteins facilitate purification and show anincreased half-life in vivo. One reported example describes chimericproteins consisting of the first two domains of the humanCD4-polypeptide and various domains of the constant regions of the heavyor light chains of mammalian immunoglobulins. (EP A 394,827; Trauneckeret al., Nature 331:84-86 (1988).) Fusion proteins havingdisulfide-linked dimeric structures (due to the IgG) can also be moreefficient in binding and neutralizing other molecules, than themonomeric secreted protein or protein fragment alone. (Fountoulakis etal., J. Biochem. 270:3958-3964 (1995).) - Similarly, EP-A-O 464 533(Canadian counterpart 2045869) discloses fusion proteins comprisingvarious portions of constant region of immunoglobulin molecules togetherwith another human protein or part thereof. In many cases, the Fc partin a fusion protein is beneficial in therapy and diagnosis, and thus canresult in, for example, improved pharmacokinetic properties. (EP-A 0232262.) Alternatively, deleting the Fc part after the fusion protein hasbeen expressed, detected, and purified, would be desired. For example,the Fc portion may hinder therapy and diagnosis if the fusion protein isused as an antigen for immunizations. In drug discovery, for example,human proteins, such as hIL-5, have been fused with Fc portions for thepurpose of high-throughput screening assays to identify antagonists ofhIL-5. (See, D. Bennett et al., J. Molecular Recognition 8:52-58 (1995);K. Johanson et al., J. Biol. Chem. 270:9459-9471 (1995).)

[0261] Moreover, the polypeptides of the present invention can be fusedto marker sequences, such as a peptide which facilitates purification ofthe fused polypeptide. In preferred embodiments, the marker amino acidsequence is a hexa-histidine peptide, such as the tag provided in a pQEvector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311),among others, many of which are commercially available. As described inGentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), forinstance, hexa-histidine provides for convenient purification of thefusion protein. Another peptide tag useful for purification, the “HA”tag, corresponds to an epitope derived from the influenza hemagglutininprotein. (Wilson et al., Cell 37:767 (1984).) Thus,. any of these abovefusions can be engineered using the polynucleotides or the polypeptidesof the present invention.

[0262] Vectors, Host Cells, and Protein Production

[0263] The present invention also relates to vectors containing thepolynucleotide of the present invention, host cells, and the productionof polypeptides by recombinant techniques. The vector may be, forexample, a phage, plasmid, viral, or retroviral vector. Retroviralvectors may be replication competent or replication defective. In thelatter case, viral propagation generally will occur only incomplementing host cells.

[0264] The polynucleotides may be joined to a vector containing aselectable marker for propagation in a host. Generally, a plasmid vectoris introduced in a precipitate, such as a calcium phosphate precipitate,or in a complex with a charged lipid. If the vector is a virus, it maybe packaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

[0265] Preferred are vectors comprising cis-acting control regions tothe polynucleotide of interest. Appropriate trans-acting factors may besupplied by the host, supplied by a complementing vector or supplied bythe vector itself upon introduction into the host.

[0266] In certain preferred embodiments in this regard, the vectorsprovide for specific expression, which may be inducible and/or celltype-specific. Particularly preferred among such vectors are thoseinducible by environmental factors that are easy to manipulate, such astemperature and nutrient additives.

[0267] Expression vectors useful in the present invention includechromosomal-, episomal- and virus-derived vectors, e.g., vectors derivedfrom bacterial plasmids, bacteriophage, yeast episomes, yeastchromosomal elements, viruses such as baculoviruses, papova viruses,vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies virusesand retroviruses, and vectors derived from combinations thereof, such ascosmids and phagemids.

[0268] The DNA insert should be operatively linked to an appropriate.Among known bacterial promoters suitable for use in the presentinvention include the E. coli lad and lacZ promoters, the T3 and T7promoters, the gpt promoter, the phage lambda PR and PL promoters, thephoA promoter and the trp promoter. Suitable eukaryotic promotersinclude the CMV immediate early promoter, the HSV thymidine kinasepromoter, the early and late SV40 promoters, the promoters of retroviralLTRs, such as those of the Rous sarcoma virus (RSV), and metallothioneinpromoters, such as the mouse metallothionein-I promoter. Other suitablepromoters will be known to the skilled artisan.

[0269] The expression constructs will further contain sites fortranscription initiation, termination and, in the transcribed region, aribosome binding site for translation. The coding portion of the maturetranscripts expressed by the constructs will preferably include atranslation initiating at the beginning and a termination codon (UAA,UGA or UAG) appropriately positioned at the end of the polypeptide to betranslated.

[0270] As indicated, the expression vectors will preferably include atleast one selectable marker. Such markers include dihydrofolatereductase, G418 or neomycin resistance for eukaryotic cell culture andtetracycline, kanamycin or ampicillin resistance genes for culturing inE. coli and other bacteria. Representative examples of appropriate hostsinclude, but are not limited to, bacterial cells, such as E. coli,Streptomyces and Salmonella typhimurium cells; fungal cells, such asyeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCCAccession No. 201178)); insect cells such as Drosophila S2 andSpodoptera Sf9 cells; animal cells such as CHO, COS, 293, and Bowesmelanoma cells; and plant cells. Appropriate culture mediums andconditions for the above-described host cells are known in the art.

[0271] Among vectors preferred for use in bacteria include pQE70, pQE60and pQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescriptvectors, pNH8A, pNH16a, pNH18A, pNH46A, available from StratageneCloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5available from Pharmacia Biotech, Inc. Among preferred eukaryoticvectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available fromStratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.Preferred expression vectors for use in yeast systems include, but arenot limited to pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ,pGAPZalph, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K, andPAO815 (all available from Invitrogen, Carlbad, Calif.). Other suitablevectors will be readily apparent to the skilled artisan.

[0272] It is specifically contemplated that the polypeptides of thepresent invention may in fact be expressed by a host cell lacking arecombinant vector.

[0273] Recombinant constructs may be introduced into host cells usingwell known techniques such as infection, transduction, transfection,transvection, electroporation and transformation. For instance,introduction of the construct into the host cell can be effected bycalcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., Basic Methods In MolecularBiology (1986).

[0274] Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes may be increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp in length that act to increasetranscriptional activity of a promoter in a given host cell-type.Examples of enhancers include the SV40 enhancer, which is located on thelate side of the replication origin at bp 100 to 270, thecytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, immunoglobulin enhancer andadenovirus enhancers.

[0275] For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide. The signals may beendogenous to the polypeptide or they may be heterologous signals.

[0276] The polypeptide may be expressed in a modified form, such as afusion protein, and may include not only secretion signals, but alsoadditional heterologous functional regions. For instance, a region ofadditional amino acids, particularly charged amino acids, may be addedto the N-terminus of the polypeptide to improve stability andpersistence in the host cell, during purification, or during subsequenthandling and storage. Also, peptide moieties may be added to thepolypeptide to facilitate purification. Such regions may be removedprior to final preparation of the polypeptide. The addition of peptidemoieties to polypeptides to engender secretion or excretion, to improvestability and to facilitate purification, among others, are familiar androutine techniques in the art. A preferred fusion protein comprises aheterologous region from immunoglobulin that is useful to stabilize andpurify proteins. For example, EP-A-0 464 533 (Canadian counterpart2045869) discloses fusion proteins comprising various portions ofconstant region of immunoglobulin molecules together with another humanprotein or part thereof. In many cases, the Fc part in a fusion proteinis thoroughly advantageous for use in therapy and diagnosis and thusresults, for example, in improved pharmacokinetic properties (EP-A 0232262). On the other hand, for some uses it would be desirable to be ableto delete the Fc part after the fusion protein has been expressed,detected and purified in the advantageous manner described. This is thecase when Fc portion proves to be a hindrance to use in therapy anddiagnosis, for example when the fusion protein is to be used as antigenfor immunizations. In drug discovery, for example, human proteins, suchas hIL-5 has been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. See,D. Bennett et al., Journal of Molecular Recognition 8:52-58 (1995) andK. et al., The Journal of Biological Chemistry 270:9459-9471 (1995).

[0277] Peptides and polypeptides of the present invention can beproduced by chemical synthetic procedures known to those of ordinaryskill in the art. For example, polypeptides up to about 80-90 amino acidresidues in length may be produced on a commercially available peptidesynthesizer model 433A (Applied Biosystems, Inc., Foster City, Calif.).Thus, as will be readily appreciated, the full-length mature BAITpolypeptide can be produced synthetically.

[0278] The BAIT protein can be recovered and purified from recombinantcell cultures by well-known methods including ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylapatite chromatographyand lectin chromatography. Most preferably, high performance liquidchromatography (“HPLC”) is employed for purification. Polypeptides ofthe present invention include naturally purified products, products ofchemical synthetic procedures, and products produced by recombinanttechniques from a prokaryotic or eukaryotic host, including, forexample, bacterial, yeast, higher plant, insect and mammalian cells.Depending upon the host employed in a recombinant production procedure,the polypeptides of the present invention may be glycosylated or may benon-glycosylated. In addition, polypeptides of the invention may alsoinclude an initial modified methionine residue, in some cases as aresult of host-mediated processes.

[0279] The polynucleotide insert should be operatively linked to anappropriate promoter, such as the phage lambda PL promoter, the E. colilac, trp, phoA and tac promoters, the SV40 early and late promoters andpromoters of retroviral LTRs, to name a few. Other suitable promoterswill be known to the skilled artisan.

[0280] Polypeptides of the present invention, and preferably thesecreted form, can also be recovered from: products purified fromnatural sources, including bodily fluids, tissues and cells, whetherdirectly isolated or cultured; products of chemical syntheticprocedures; and products produced by recombinant techniques from aprokaryotic or eukaryotic host, including, for example, bacterial,yeast, higher plant, insect, and mammalian cells. Depending upon thehost employed in a recombinant production procedure, the polypeptides ofthe present invention may be glycosylated or may be non-glycosylated. Inaddition, polypeptides of the invention may also include an initialmodified methionine residue, in some cases as a result of host-mediatedprocesses. Thus, it is well known in the art that the N-terminalmethionine encoded by the translation initiation codon generally isremoved with high efficiency from any protein after translation in alleukaryotic cells. While the N-terminal methionine on most proteins alsois efficiently removed in most prokaryotes, for some proteins, thisprokaryotic removal process is inefficient, depending on the nature ofthe amino acid to which the N-terminal methionine is covalently linked.

[0281] In one embodiment, the yeast Pichia pastoris is used to expressthe polypeptide of the present invention in a eukaryotic system. Pichiapastoris is a methylotrophic yeast which can metabolize methanol as itssole carbon source. A main step in the methanol metabolization pathwayis the oxidation of methanol to formaldehyde using O₂. This reaction iscatalyzed by the enzyme alcohol oxidase. In order to metabolize methanolas its sole carbon source, Pichia pastoris must generate high levels ofalcohol oxidase due, in part, to the relatively low affinity of alcoholoxidase for O₂. Consequently, in a growth medium depending on methanolas a main carbon source, the promoter region of one of the two alcoholoxidase genes (AOX1) is highly active. In the presence of methanol,alcohol oxidase produced from the AOX1 gene comprises up toapproximately 30% of the total soluble protein in Pichia pastoris. See,Ellis, S. B., et al., Mol. Cell. Biol. 5:1111-21 (1985); Koutz, P. J, etal., Yeast 5:167-77 (1989); Tschopp, J. F., et al., Nucl. Acids Res.15:3859-76 (1987). Thus, a heterologous coding sequence, such as, forexample, a polynucleotide of the present invention, under thetranscriptional regulation of all or part of the AOX1 regulatorysequence is expressed at exceptionally high levels in Pichia yeast grownin the presence of methanol.

[0282] In one example, the plasmid vector pPIC9K is used to express DNAencoding a polypeptide of the invention, as set forth herein, in aPichea yeast system essentially as described in “Pichia Protocols:Methods in Molecular Biology,” D. R. Higgins and J. Cregg, eds. TheHumana Press, Totowa, N.J., 1998. This expression vector allowsexpression and secretion of a protein of the invention by virtue of thestrong AOX1 promoter linked to the Pichia pastoris alkaline phosphatase(PHO) secretory signal peptide (i.e., leader) located upstream of amultiple cloning site.

[0283] Many other yeast vectors could be used in place of pPIC9K, suchas, pYES2, pYD1, pTEF1I/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9,pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, and PAO815, as one skilled in theart would readily appreciate, as long as the proposed expressionconstruct provides appropriately located signals for transcription,translation, secretion (if desired), and the like, including an in-frameAUG as required.

[0284] In another embodiment, high-level expression of a heterologouscoding sequence, such as, for example, a polynucleotide of the presentinvention, may be achieved by cloning the heterologous polynucleotide ofthe invention into an expression vector such as, for example, pGAPZ orpGAPZalpha, and growing the yeast culture in the absence of methanol.

[0285] In addition to encompassing host cells containing the vectorconstructs discussed herein, the invention also encompasses primary,secondary, and immortalized host cells of vertebrate origin,particularly mammalian origin, that have been engineered to delete orreplace endogenous genetic material (e.g., coding sequence), and/or toinclude genetic material (e.g., heterologous polynucleotide sequences)that is operably associated with the polynucleotides of the invention,and which activates, alters, and/or amplifies endogenouspolynucleotides. For example, techniques known in the art may be used tooperably associate heterologous control regions (e.g., promoter and/orenhancer) and endogenous polynucleotide sequences via homologousrecombination, resulting in the formation of a new transcription unit(see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; U.S. Pat. No.5,733,761, issued Mar. 31, 1998; International Publication No. WO96/29411, published Sep. 26, 1996; International Publication No. WO94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci.USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989),the disclosures of each of which are incorporated by reference in theirentireties).

[0286] In addition, polypeptides of the invention can be chemicallysynthesized using techniques known in the art (e.g., see Creighton,1983, Proteins: Structures and Molecular Principles, W.H. Freeman & Co.,N.Y., and Hunkapiller et al., Nature, 310:105-111 (1984)). For example,a polypeptide corresponding to a fragment of a polypeptide sequence ofthe invention can be synthesized by use of a peptide synthesizer.Furthermore, if desired, nonclassical amino acids or chemical amino acidanalogs can be introduced as a substitution or addition into thepolypeptide sequence. Non-classical amino acids include, but are notlimited to, to the D-isomers of the common amino acids,2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid,Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib,2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine,norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline,cysteic acid, t-butylglycine, t- butylalanine, phenylglycine,cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acidssuch as b-methyl amino acids, Ca-methyl amino acids, Na-methyl aminoacids, and amino acid analogs in general. Furthermore, the amino acidcan be D (dextrorotary) or L (levorotary).

[0287] The invention encompasses polypeptides which are differentiallymodified during or after translation, e.g., by glycosylation,acetylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to an antibodymolecule or other cellular ligand, etc. Any of numerous chemicalmodifications may be carried out by known techniques, including but notlimited, to specific chemical cleavage by cyanogen bromide, trypsin,chymotrypsin, papain, V8 protease, NaBH₄; acetylation, formylation,oxidation, reduction; metabolic synthesis in the presence oftunicamycin; etc.

[0288] Additional post-translational modifications encompassed by theinvention include, for example, e.g., N-linked or O-linked carbohydratechains, processing of N-terminal or C-terminal ends), attachment ofchemical moieties to the amino acid backbone, chemical modifications ofN-linked or O-linked carbohydrate chains, and addition or deletion of anN-terminal methionine residue as a result of procaryotic host cellexpression. The polypeptides may also be modified with a detectablelabel, such as an enzymatic, fluorescent, isotopic or affinity label toallow for detection and isolation of the protein.

[0289] Also provided by the invention are chemically modifiedderivatives of the polypeptides of the invention which may provideadditional advantages such as increased solubility, stability andcirculating time of the polypeptide, or decreased immunogenicity (seeU.S. Pat. No. 4,179,337). The chemical moieties for derivitization maybe selected from water soluble polymers such as polyethylene glycol,ethylene glycol/propylene glycol copolymers, carboxymethylcellulose,dextran, polyvinyl alcohol and the like. The polypeptides may bemodified at random positions within the molecule, or at predeterminedpositions within the molecule and may include one, two, three or moreattached chemical moieties.

[0290] The polymer may be of any molecular weight, and may be branchedor unbranched. For polyethylene glycol, the preferred molecular weightis between about 1 kDa and about 100 kDa (the term “about” indicatingthat in preparations of polyethylene glycol, some molecules will weighmore, some less, than the stated molecular weight) for ease in handlingand manufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog).

[0291] The polyethylene glycol molecules (or other chemical moieties)should be attached to the protein with consideration of effects onfunctional or antigenic domains of the protein. There are a number ofattachment methods available to those skilled in the art, e.g., EP 0 401384, herein incorporated by reference (coupling PEG to G-CSF), see alsoMalik et al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation ofGM-CSF using tresyl chloride). For example, polyethylene glycol may becovalently bound through amino acid residues via a reactive group, suchas, a free amino or carboxyl group. Reactive groups are those to whichan activated polyethylene glycol molecule may be bound. The amino acidresidues having a free amino group may include lysine residues and theN-terminal amino acid residues; those having a free carboxyl group mayinclude aspartic acid residues glutamic acid residues and the C-terminalamino acid residue. Sulfhydryl groups may also be used as a reactivegroup for attaching the polyethylene glycol molecules. Preferred fortherapeutic purposes is attachment at an amino group, such as attachmentat the N-terminus or lysine group.

[0292] One may specifically desire proteins chemically modified at theN-terminus. Using polyethylene glycol as an illustration of the presentcomposition, one may select from a variety of polyethylene glycolmolecules (by molecular weight, branching, etc.), the proportion ofpolyethylene glycol molecules to protein (polypeptide) molecules in thereaction mix, the type of pegylation reaction to be performed, and themethod of obtaining the selected N-terminally pegylated protein. Themethod of obtaining the N-terminally pegylated preparation (i.e.,separating this moiety from other monopegylated moieties if necessary)may be by purification of the N-terminally pegylated material from apopulation of pegylated protein molecules. Selective proteins chemicallymodified at the N-terminus modification may be accomplished by reductivealkylation which exploits differential reactivity of different types ofprimary amino groups (lysine versus the N-terminal) available forderivatization in a particular protein. Under the appropriate reactionconditions, substantially selective derivatization of the protein at theN-terminus with a carbonyl group containing polymer is achieved.

[0293] The polypeptides of the invention may be in monomers or multimers(i.e., dimers, trimers, tetramers and higher multimers). Accordingly,the present invention relates to monomers and multimers of thepolypeptides of the invention, their preparation, and compositions(preferably, Therapeutics) containing them. In specific embodiments, thepolypeptides of the invention are monomers, dimers, trimers ortetramers. In additional embodiments, the multimers of the invention areat least dimers, at least trimers, or at least tetramers.

[0294] Multimers encompassed by the invention may be homomers orheteromers. As used herein, the term homomer, refers to a multimercontaining only polypeptides corresponding to the amino acid sequence ofSEQ ID NO:Y or encoded by the cDNA contained in a deposited clone(including fragments, variants, splice variants, and fusion proteins,corresponding to these polypeptides as described herein). These homomersmay contain polypeptides having identical or different amino acidsequences. In a specific embodiment, a homomer of the invention is amultimer containing only polypeptides having an identical amino acidsequence. In another specific embodiment, a homomer of the invention isa multimer containing polypeptides having different amino acidsequences. In specific embodiments, the multimer of the invention is ahomodimer (e.g., containing polypeptides having identical or differentamino acid sequences) or a homotrimer (e.g., containing polypeptideshaving identical and/or different amino acid sequences). In additionalembodiments, the homomeric multimer of the invention is at least ahomodimer, at least a homotrimer, or at least a homotetramer.

[0295] As used herein, the term heteromer refers to a multimercontaining one or more heterologous polypeptides (i.e., polypeptides ofdifferent proteins) in addition to the polypeptides of the invention. Ina specific embodiment, the multimer of the invention is a heterodimer, aheterotrimer, or a heterotetramer. In additional embodiments, theheteromeric multimer of the invention is at least a heterodimer, atleast a heterotrimer, or at least a heterotetramer.

[0296] Multimers of the invention may be the result of hydrophobic,hydrophilic, ionic and/or covalent associations and/or may be indirectlylinked, by for example, liposome formation. Thus, in one embodiment,multimers of the invention, such as, for example, homodimers orhomotrimers, are formed when polypeptides of the invention contact oneanother in solution. In another embodiment, heteromultimers of theinvention, such as, for example, heterotrimers or heterotetramers, areformed when polypeptides of the invention contact antibodies to thepolypeptides of the invention (including antibodies to the heterologouspolypeptide sequence in a fusion protein of the invention) in solution.In other embodiments, multimers of the invention are formed by covalentassociations with and/or between the polypeptides of the invention. Suchcovalent associations may involve one or more amino acid residuescontained in the polypeptide sequence (e.g., that recited in thesequence listing, or contained in the polypeptide encoded by a depositedclone). In one instance, the covalent associations are cross-linkingbetween cysteine residues located within the polypeptide sequences whichinteract in the native (i.e., naturally occurring) polypeptide. Inanother instance, the covalent associations are the consequence ofchemical or recombinant manipulation. Alternatively, such covalentassociations may involve one or more amino acid residues contained inthe heterologous polypeptide sequence in a fusion protein of theinvention.

[0297] In one example, covalent associations are between theheterologous sequence contained in a fusion protein of the invention(see, e.g., U.S. Pat. No. 5,478,925). In a specific example, thecovalent associations are between the heterologous sequence contained inan Fc fusion protein of the invention (as described herein). In anotherspecific example, covalent associations of fusion proteins of theinvention are between heterologous polypeptide sequence from anotherprotein that is capable of forming covalently associated multimers, suchas for example, oseteoprotegerin (see, e.g., International PublicationNO: WO 98/49305, the contents of which are herein incorporated byreference in its entirety). In another embodiment, two or morepolypeptides of the invention are joined through peptide linkers.Examples include those peptide linkers described in U.S. Pat. No.5,073,627 (hereby incorporated by reference). Proteins comprisingmultiple polypeptides of the invention separated by peptide linkers maybe produced using conventional recombinant DNA technology.

[0298] Another method for preparing multimer polypeptides of theinvention involves use of polypeptides of the invention fused to aleucine zipper or isoleucine zipper polypeptide sequence. Leucine zipperand isoleucine zipper domains are polypeptides that promotemultimerization of the proteins in which they are found. Leucine zipperswere originally identified in several DNA-binding proteins (Landschulzet al., Science 240:1759, (1988)), and have since been found in avariety of different proteins. Among the known leucine zippers arenaturally occurring peptides and derivatives thereof that dimerize ortrimerize. Examples of leucine zipper domains suitable for producingsoluble multimeric proteins of the invention are those described in PCTapplication WO 94/10308, hereby incorporated by reference. Recombinantfusion proteins comprising a polypeptide of the invention fused to apolypeptide sequence that dimerizes or trimerizes in solution areexpressed in suitable host cells, and the resulting soluble multimericfusion protein is recovered from the culture supernatant usingtechniques known in the art.

[0299] Trimeric polypeptides of the invention may offer the advantage ofenhanced biological activity. Preferred leucine zipper moieties andisoleucine moieties are those that preferentially form trimers. Oneexample is a leucine zipper derived from lung surfactant protein D(SPD), as described in Hoppe et al. (FEBS Letters 344:191, (1994)) andin U.S. patent application Ser. No. 08/446,922, hereby incorporated byreference. Other peptides derived from naturally occurring trimericproteins may be employed in preparing trimeric polypeptides of theinvention.

[0300] In another example, proteins of the invention are associated byinteractions between Flag® polypeptide sequence contained in fusionproteins of the invention containing Flag® polypeptide seuqence. In afurther embodiment, associations proteins of the invention areassociated by interactions between heterologous polypeptide sequencecontained in Flag® fusion proteins of the invention and anti-Flag®antibody.

[0301] The multimers of the invention may be generated using chemicaltechniques known in the art. For example, polypeptides desired to becontained in the multimers of the invention may be chemicallycross-linked using linker molecules and linker molecule lengthoptimization techniques known in the art (see, e.g., U.S. Pat. No.5,478,925, which is herein incorporated by reference in its entirety).Additionally, multimers of the invention may be generated usingtechniques known in the art to form one or more inter-moleculecross-links between the cysteine residues located within the sequence ofthe polypeptides desired to be contained in the multimer (see, e.g.,U.S. Pat. No. 5,478,925, which is herein incorporated by reference inits entirety). Further, polypeptides of the invention may be routinelymodified by the addition of cysteine or biotin to the C terminus orN-terminus of the polypeptide and techniques known in the art may beapplied to generate multimers containing one or more of these modifiedpolypeptides (see, e.g., U.S. Pat. No. 5,478,925, which is hereinincorporated by reference in its entirety). Additionally, techniquesknown in the art may be applied to generate liposomes containing thepolypeptide components desired to be contained in the multimer of theinvention (see, e.g., U.S. Pat. No. 5,478,925, which is hereinincorporated by reference in its entirety).

[0302] Alternatively, multimers of the invention may be generated usinggenetic engineering techniques known in the art. In one embodiment,polypeptides contained in multimers of the invention are producedrecombinantly using fusion protein technology described herein orotherwise known in the art (see, e.g., U.S. Pat. No. 5,478,925, which isherein incorporated by reference in its entirety). In a specificembodiment, polynucleotides coding for a homodimer of the invention aregenerated by ligating a polynucleotide sequence encoding a polypeptideof the invention to a sequence encoding a linker polypeptide and thenfurther to a synthetic polynucleotide encoding the translated product ofthe polypeptide in the reverse orientation from the original C-terminusto the N-terminus (lacking the leader sequence) (see, e.g., U.S. Pat.No. 5,478,925, which is herein incorporated by reference in itsentirety). In another embodiment, recombinant techniques describedherein or otherwise known in the art are applied to generate recombinantpolypeptides of the invention which contain a transmembrane domain (orhyrophobic or signal peptide) and which can be incorporated by membranereconstitution techniques into liposomes (see, e.g., U.S. Pat. No.5,478,925, which is herein incorporated by reference in its entirety).

[0303] Diagnosis of Nervous System-Related Disorders

[0304] The present inventors have discovered that BAIT is expressed inwhole human brain, and to a much lesser extent in adult pancreas andadult heart. More particularly, by Northern blotting a 2 kb MRNA wasexpressed mostly in adult brain (at a relative level of ˜5×) and to amuch lesser extent in adult pancreas (˜1×) and adult heart (˜0.5×).Adult tissues not expressing significant amounts of mRNA includeplacenta, lung, liver, skeletal muscle, kidney, spleen, thymus,prostate, testis, ovary, small intestine, colon, and peripheral bloodleukocytes. In addition, in the nervous system a 2 kb mRNA was seen incerebral cortex, medulla, occipital lobe, frontal lobe, temporal lobe,putamen, and spinal cord but not in cerebellum. In the chicken,neuroserpin, the presumptive ortholog of the human BAIT protein, wasfound to be secreted from axons of both CNS and PNS neurons. Osterwalderet al., supra. The most prominent expression of neuroserpin in adultchickens is found in the hyperstriatum accessorium, the neostriaum andthe hippocampus, plastic regions of the adult brain involved inprocesses of learning and memory where a subtle balance between andanti-proteolytic activities seems to be required for appropriatesynaptic function. Id. at 295 1. Further, transgenic mice with anenhanced proteolytic activity in the cortex and hippocampus due tooverexpression of urokinase-type plasminogen activator (u-PA) have beenfound to exhibit impaired spatial, olfactory and tasteaversion learning.Id. Further still, elimination of a serpin inhibitor of u-PA, PNI(described above) by homologous recombination leads to reduced long-termpotentiation (LTP) of learning, whereas overexpression of PNI results inenhanced LTP of hippocampal neurons. Id. The available observations ontemporal-spatial patterns of expression of neuroserpin the chicken andBAIT polypeptide in human tissues implicate BAIT as a regulator forsynaptogenesis and the subsequent remodelling processes includingsynapse elimination rather than neurite outgrowth. Id.

[0305] Accordingly, for a number of disorders of the central orperipheral nervous system, significantly higher or lower levels of BAITgene expression may be detected in certain tissues (e.g., adult brain,embryonic retina, cerebellum and spinal chord), or bodily fluids (e.g.,serum, plasma, urine, synovial fluid or spinal fluid) taken from anindividual having such a disorder, relative to a “standard” BAIT geneexpression level, i.e., the BAIT expression level in healthy tissue froman individual not having the nervous system disorder. Thus, theinvention provides a diagnostic method useful during diagnosis ofnervous system disorders, which involves: (a) assaying BAIT geneexpression level in cells or body fluid of an individual; (b) comparingthe BAIT gene expression level with a standard BAIT gene expressionlevel, whereby an increase or decrease in the assayed BAIT geneexpression level compared to the standard expression level is indicativeof disorder in the nervous system.

[0306] By individual is intended mammalian individuals, preferablyhumans, including adults, children, babies and embryos or fetuses at allstages of development of the nervous system. By “measuring theexpression level of the gene encoding the BAIT protein” is intendedqualitatively or quantitatively measuring or estimating the level of theBAIT protein or the level of the mRNA encoding the BAIT protein in afirst biological sample either directly (e.g., by determining orestimating absolute protein level or mRINA level) or relatively (e.g.,by comparing to the BAIT protein level or mRNA level in a secondbiological sample). Preferably, the BAIT protein level or mRNA level inthe first biological sample is measured or estimated and compared to astandard BAIT protein level or mRNA level, the standard being taken froma second biological sample obtained from an individual not having thedisorder or being determined by averaging levels from a population ofindividuals not having a disorder of the immune system. As will beappreciated in the art, once a standard BAIT protein level or mRNA levelis known, it can be used repeatedly as a standard for comparison.

[0307] By “biological sample” is intended any biological sample obtainedfrom an individual, body fluid, cell line, tissue culture, or othersource which contains BAIT protein or mRNA. As indicated, biologicalsamples include body fluids (such as sera, plasma, urine, synovial fluidand spinal fluid) which contain secreted mature BAIT protein, nervoussystem tissue, and other tissue sources found to express BAIT or a BAITreceptor. Methods for obtaining tissue biopsies and body fluids frommammals are well known in the art. Where the biological sample is toinclude mRNA, a tissue biopsy is the preferred source.

[0308] The present invention is usetul for diagnosis of various nervoussystem-related disorders in mammals, preferably humans. Such disordersinclude impaired processes of learning and memory, including impairedspatial, olfactory and taste aversion learning, learning and memoryimpairments associated with Alzheimer's disease, and the like.

[0309] Total cellular RNA can be isolated from a biological sample usingany suitable technique such as the single-stepguanidinium-thiocyanate-phenol-chloroform method described inChomczynski and Sacchi, Anal. Biochem. 162:156-159 (1987). Levels ofmRNA encoding the BAIT protein are then assayed using any appropriatemethod. These include Northern blot analysis, SI nuclease mapping, thepolymerase chain reaction (PCR), reverse transcription in combinationwith the polymerase chain reaction (RT-PCR), and reverse transcriptionin combination with the ligase chain reaction (RT-LCR).

[0310] Northern blot analysis can be performed as described in Harada etal., Cell 63:303-312 (1990). Briefly, total RNA is prepared from abiological sample as described above. For the Northern blot, the RNA isdenatured in an appropriate buffer (such as glyoxal/dimethylsulfoxide/sodium phosphate buffer), subjected to agarose gelelectrophoresis, and transferred onto a nitrocellulose filter. After theRNAs have been linked to the filter by a UV linker, the filter isprehybridized in a solution containing formamide, SSC, Denhardt'ssolution, denatured salmon sperm, SDS, and sodium phosphate buffer. BAITprotein cDNA labeled according to any appropriate method (such as the³²P-multiprimed DNA labeling system (Amersham)) is used as probe. Afterhybridization overnight, the filter is washed and exposed to x-ray film.cDNA for use as probe according to the present invention is described inthe sections above and will preferably be at least 15 bp in length.

[0311] S1 mapping can be performed as described in Fujita et al., Cell49:357-367 (1987). To prepare probe DNA for use in S1 mapping, the sensestrand of above-described cDNA is used as a template to synthesizelabeled antisense DNA. The antisense DNA can then be digested using anappropriate restriction endonuclease to generate further DNA probes of adesired length. Such antisense probes are useful for visualizingprotected bands corresponding to the target mRNA (i.e., mRNA encodingthe BAIT protein). Northern blot analysis can be performed as describedabove.

[0312] Preferably, levels of mRNA encoding the BAIT protein are assayedusing the RT-PCR method described in Makino et al., Technique 2:295-301(1990). By this method, the radioactivities of the “amplicons” in thepolyacrylamide gel bands are linearly related to the initialconcentration of the target mRNA. Briefly, this method involves addingtotal RNA isolated from a biological sample in a reaction mixturecontaining a RT primer and appropriate buffer. After incubating forprimer annealing, the mixture can be supplemented with a RT buffer,dNTPs, DTT, RNase inhibitor and reverse transcriptase. After incubationto achieve reverse transcription of the RNA, the RT products are thensubject to PCR using labeled primers. Alternatively, rather thanlabeling the primers, a labeled dNTP can be included in the PCR reactionmixture. PCR amplification can be performed in a DNA thermal cycleraccording to conventional techniques. After a suitable number of roundsto achieve amplification, the PCR reaction mixture is electrophoresed ona polyacrylamide gel. After drying the gel, the radioactivity of theappropriate bands (corresponding to the mRNA encoding the BAIT protein))is quantified using an imaging analyzer. RT and PCR reaction ingredientsand conditions, reagent and gel concentrations, and labeling methods arewell known in the art. Variations on the RT-PCR method will be apparentto the skilled artisan. Any set of oligonucleotide primers which willamplify reverse transcribed target mRNA can be used and can be designedas described in the sections above.

[0313] Assaying BAIT protein levels in a biological sample can occurusing any art-known method. Preferred for assaying BAIT protein levelsin a biological sample are antibody-based techniques. For example, BAITprotein expression in tissues can be studied with classicalimmunohistological methods. In these, the specific recognition isprovided by the primary antibody (polyclonal or monoclonal) but thesecondary detection system can utilize fluorescent, enzyme, or otherconjugated secondary antibodies. As a result, an immunohistologicalstaining of tissue section for pathological examination is obtained.Tissues can also be extracted, e.g., with urea and neutral detergent,for the liberation of BAIT protein for Western-blot or dot/slot assay(Jalkanen, M., et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, M.,et al., J. Cell Biol. 105:3087-3096 (1987)). In this technique, which isbased on the use of cationic solid phases, quantitation of BAIT proteincan be accomplished using isolated BAIT protein as a standard. Thistechnique can also be applied to body fluids. With these samples, amolar concentration of BAIT protein will aid to set standard values ofBAIT protein content for different body fluids, like serum, plasma,urine, spinal fluid, etc. The normal appearance of BAIT protein amountscan then be set using values from healthy individuals, which can becompared to those obtained from a test subject.

[0314] Other antibody-based methods useful for detecting BAIT proteingene expression include immunoassays, such as the enzyme linkedimmunosorbent assay (ELISA) and the radioimmunoassay (RIA). For example,a BAIT protein-specific monoclonal antibody can be used both as animmunoadsorbent and as an enzyme-labeled probe to detect and quantifythe BAIT protein. The amount of BAIT protein present in the sample canbe calculated by reference to the amount present in a standardpreparation using a linear regression computer algorithm. Such an ELISAfor detecting a tumor antigen is described in lacobelli et al., BreastCancer Research and Treatment 11: 19-30 (1988). In another ELISA assay,two distinct specific monoclonal antibodies can be used to detect BAITprotein in a body fluid. In this assay, one of the antibodies is used asthe immunoadsorbent and the other as the enzyme-labeled probe.

[0315] The above techniques may be conducted essentially as a “one-step”or “two-step” assay. The “one-step” assay involves contacting BAITprotein with immobilized antibody and, without washing, contacting themixture with the labeled antibody. The “two-step” assay involves washingbefore contacting the mixture with the labeled antibody. Otherconventional methods may also be employed as suitable. It is usuallydesirable to immobilize one component of the assay system on a support,thereby allowing other components of the system to be brought intocontact with the component and readily removed from the sample.

[0316] Suitable enzyme labels include, for example, those from theoxidase group, which catalyze the production of hydrogen peroxide byreacting with substrate. Glucose oxidase is particularly preferred as ithas good stability and its substrate (glucose) is readily available.Activity of an oxidase label may be assayed by measuring theconcentration of hydrogen peroxide formed by the enzyme-labeledantibody/substrate reaction. Besides enzymes, other suitable labelsinclude radioisotopes, such as iodine (¹²⁵I, ¹²¹I), carbon (¹⁴C), sulfur(³⁵S), tritium (³H), indium (¹¹²In), and technetium (⁹⁹mTc), andfluorescent labels, such as fluorescein and rhodamine, and biotin Inaddition to assaying BAIT protein levels in a biological sample obtainedfrom an individual, BAIT protein can also be detected in vivo byimaging. Antibody labels or markers for in vivo imaging of BAIT proteininclude those detectable by X-radiography, NMR or ESR. ForX-radiography, suitable labels include radioisotopes such as barium orcesium, which emit detectable radiation but are not overtly harmful tothe subject. Suitable markers for NMR and ESR include those with adetectable characteristic spin, such as deuterium, which may beincorporated into the antibody by labeling of nutrients for the relevanthybridoma.

[0317] A BAIT protein-specific antibody or antibody fragment which hasbeen labeled with an appropriate detectable imaging moiety, such as aradioisotope (for example, ¹³¹I, ¹¹²In, ^(99m)Tc), a radio-opaquesubstance, or a material detectable by nuclear magnetic resonance, isintroduced (for example, parenterally, subcutaneously orintraperitoneally) into the mammal to be examined for immune systemdisorder. It will be understood in the art that the size of the subjectand the imaging system used will determine the quantity of imagingmoiety needed to produce diagnostic images. In the case of aradioisotope moiety, for a human subject, the quantity of radioactivityinjected will normally range from about 5 to 20 millicuries of ^(99m)Tc.The labeled antibody or antibody fragment will then preferentiallyaccumulate at the location of cells which contain BAIT protein. In vivotumor imaging is described in S. W. Burchiel et al.,“Immunopharmaco-kinetics of Radiolabeled Antibodies and Their Fragments”(Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982)).

[0318] BAIT-protein specific antibodies for use in the present inventioncan be raised against the intact BAIT protein or an antigenicpolypeptide fragment thereof, which may be presented together with acarrier protein, such as an albumin, to an animal system (such as rabbitor mouse) or, if it is long enough (at least about 25 amino acids),without a carrier.

[0319] As used herein, the term “antibody” (Ab) or “monoclonal antibody”(Mab) is meant to include intact molecules as well as antibody fragments(such as, for example, Fab and F(ab′)₂ fragments) which are capable ofspecifically binding to BAIT protein. Fab and F(ab′)₂ fragments lack theFc fragment of intact antibody, clear more rapidly from the circulation,and may have less non-specific tissue binding of an intact antibody(Wahl et al., J. Nucl. Med. 24.316-325 (1983)). Thus, these fragmentsare preferred.

[0320] The antibodies of the present invention may be prepared by any ofa variety of methods. For example, cells expressing the BAIT protein oran antigenic fragment thereof can be administered to an animal in orderto induce the production of sera containing polyclonal antibodies. In apreferred method, a preparation of BAIT protein is prepared and purifiedto render it substantially free of natural contaminants. Such apreparation is then introduced into an animal in order to producepolyclonal antisera of greater specific activity.

[0321] In the most preferred method, the antibodies of the presentinvention are monoclonal antibodies (or BAIT protein binding fragmentsthereof). Such monoclonal antibodies can be prepared using hybridomatechnology (Kohler et al. Nature 256:495 (1975); Kohler et al., Eur. J.Immunol. 6:511 (1976); Kohler et al., Eur. J. Immunol. 6:292 (1976);Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas,Elsevier, N.Y., (1981) pp. 563-681). In general, such procedures involveimmunizing an animal (preferably a mouse) with a BAIT protein antigenor, more preferably, with a BAIT protein-expressing cell. Suitable cellscan be recognized by their capacity to bind anti-BAIT protein antibody.Such cells may be cultured in any suitable tissue culture medium;however, it is preferable to culture cells in Earle's modified Eagle'smedium supplemented with 10% fetal bovine serum (inactivated at about56° C.), and supplemented with about 10 g/l of nonessential amino acids,about 1,000 U/ml of penicillin, and about 100 g/ml of streptomycin. Thesplenocytes of such mice are extracted and fused with a suitable myelomacell line. Any suitable myeloma cell line may be employed in accordancewith the present invention; however, it is preferable to employ theparent myeloma cell line (SP₂O), available from the American TypeCulture Collection, Manassas, Va. After fusion, the resulting hybridomacells are selectively maintained in HAT medium, and then cloned bylimiting dilution as described by Wands et al. (Gastroenterology80:225-232 (198 1)). The hybridoma cells obtained through such aselection are then assayed to identify clones which secrete antibodiescapable of binding the BAIT protein antigen.

[0322] Alternatively, additional antibodies capable of binding to theBAIT protein antigen may be produced in a two-step procedure through theuse of anti-idiotypic antibodies. Such a method makes use of the factthat antibodies are themselves antigens, and that, therefore, it ispossible to obtain an antibody which binds to a second antibody. Inaccordance with this method, BAIT-protein specific antibodies are usedto immunize an animal, preferably a mouse. The splenocytes of such ananimal are then used to produce hybridoma cells, and the hybridoma cellsare screened to identify clones which produce an antibody whose abilityto bind to the BAIT protein-specific antibody can be blocked by the BAITprotein antigen. Such antibodies comprise anti-idiotypic antibodies tothe BAIT protein-specific antibody and can be used to immunize an animalto induce formation of further BAIT protein-specific antibodies.

[0323] It will be appreciated that Fab and F(ab′)₂ and other fragmentsof the antibodies of the present invention may be used according to themethods disclosed herein. Such fragments are typically produced byproteolytic cleavage, using enzymes such as papain (to produce Fabfragments) or pepsin (to produce F(ab′)₂ fragments). Alternatively, BAITprotein-binding fragments can be produced through the application ofrecombinant DNA technology or through synthetic chemistry.

[0324] Where in vivo imaging is used to detect enhanced levels of BAITprotein for diagnosis in humans, it may be preferable to use “humanized”chimeric monoclonal antibodies. Such antibodies can be produced usinggenetic constructs derived from hybridoma cells producing the monoclonalantibodies described above. Methods for producing chimeric antibodiesare known in the art. See, for review, Morrison, Science 229:1202(1985); Oi et al., BioTechniques 4:214 (1986); Cabilly et al., U.S. Pat.No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494;Neuberger et al., WO 8601533; Robinson et al., WO 8702671; Boulianne etal., Nature 312:643 (1984); Neuberger et al., Nature 314:268 (1985).

[0325] Further suitable labels for the BAIT protein-specific antibodiesof the present invention are provided below. Examples of suitable enzymelabels include malate dehydrogenase, staphylococcal nuclease,delta-5-steroid isomerase, yeast-alcohol dehydrogenase, alpha-glycerolphosphate dehydrogenase, triose phosphate isomerase, peroxidase,alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphatedehydrogenase, glucoamylase, and acetylcholine esterase.

[0326] Examples of suitable radioisotopic labels include ³H, ¹¹¹In,¹²⁵I, ¹³¹I, ³²P, ³⁵S, ¹⁴C, ⁵¹Cr, ⁵⁷To, ⁵⁸Co, ⁵⁹Fe, ⁷⁵Se, ¹⁵²Eu, ⁹⁰Y,⁶⁷Cu, ²¹⁷Ci, ²¹¹At, ²¹²Pb, ⁴⁷Sc, ¹⁰⁹Pd, etc. ¹¹¹In is a preferredisotope where in vivo imaging is used since it avoids the problem ofdehalogenation of the ¹²⁵I or ¹³¹I-labeled monoclonal antibody by theliver. In addition, this radionucleotide has a more favorable gammaemission energy for imaging (Perkins et al., Eur. J. Nucl. Med.10:296-301 (1985); Carasquillo et al., J. Nucl. Med. 28:281-287 (1987)).For example, “‘In coupled to monoclonal antibodies with1-(P-isothiocyanatobenzyl)-DPTA has shown little uptake in non-tumoroustissues, particularly the liver, and therefore enhances specificity oftumor localization (Esteban et al., J. Nucl. Med. 28:861-870 (1987)).Examples of suitable non-radioactive isotopic labels include ¹⁵⁷Gd,⁵⁵Mn, ¹⁶²Dy, ⁵²Tr, and ⁵⁶Fe.

[0327] Examples of suitable fluorescent labels include an ¹¹²Eu label, afluorescein label, an isothiocyanate label, a rhodamine label, aphycoerythrin label, a phycocyanin label, an allophycocyanin label, anophthaldehyde label, and a fluorescamine label. Examples of suitabletoxin labels include diphtheria toxin, ricin, and cholera toxin.Examples of chemiluminescent labels include a luminal label, anisoluminal label, an aromatic acridinium ester label, an imidazolelabel, an acridinium salt label, an oxalate ester label, a luciferinlabel, a luciferase label, and an aequorin label. Examples of nuclearmagnetic resonance contrasting agents include heavy metal nuclei such asGd, Mn, and iron.

[0328] Typical techniques for binding the above-described labels toantibodies are provided by Kennedy et al., Clin. Chim. Acta 70:1-31(1976), and Schurs et al., Clin. Chim. Acta 81:1-40 (1977). Couplingtechniques mentioned in the latter are the glutaraldehyde method, theperiodate method, the dimaleimide method, them-maleimidobenzyl-N-hydroxy-succinimide ester method, all of whichmethods are incorporated by reference herein.

[0329] Treatment of Nervous System-Related and Other Disorders

[0330] As noted above, BAIT polynucleotides, polypeptides and otheraspects of this invention are useful for diagnosis of various nervoussystem-related disorders in mammals, including impaired processes oflearning and memory, including impaired spatial, olfactory andtaste-aversion learning, learning and memory impairments associated withAlzheimer's disease, and the like. Given the activities modulated byBAIT, it is readily apparent that a substantially altered (increased ordecreased) level of expression of BAIT in an individual compared to thestandard or “normal” level produces pathological conditions such asthose described above in relation to diagnosis of nervous system-relateddisorders. It will also be appreciated by one of ordinary skill that,since the BAIT protein of the invention is translated with a leaderpeptide suitable for secretion of the mature protein from the cellswhich express BAIT, when BAIT protein (particularly the mature form) isadded from an exogenous source to cells, tissues or the body of anindividual, the protein will exert its modulating activities on any ofits target cells of that individual. Therefore, it will be appreciatedthat conditions caused by a decrease in the standard or normal level ofBAIT activity in an individual, or an increase in a protease susceptibleto inhibition by BAIT, particularly disorders of the nervous system, canbe treated by administration of BAIT protein.

[0331] The human BAIT protein of the present invention has been shown toexhibit selective inhibition of tissue-type plasminogen activator (t-PA)with a lesser degree of inhibition of trypsin, thrombin orurokinase-type plasminogen activator (u-PA). More in particular, invitro enzymatic activity has been demonstrated for the baculovirusexpressed purified protein. FIG. 5 shows the inhibition of t-PA, u-PA,plasmin, trypsin, and thrombin proteolytic activity with increasingamounts of purified protein expressed and purified as described below.t-PA was inhibited with a half-maximal inhibitory concentration IC₅₀) of200 nM, u-PA and trypsin were inhibited at an IC₅₀ of 1 μM and 0.7 μM,respectively. No other protease was inhibited to 50% of control. Therate constant for BAIT reaction with tPA is about 7.8±1.5×10⁴ mol/sec.

[0332] More in particular, the inhibitory activity against various tPA(Genentech), uPA (Serono), plasmin (a gift of Dr. D. Strickland),thrombin (a gift of Dr. S. T. Olson), and β-trypsin (a gift of Dr. S. T.Olson), was determined in a single step chromogenic assay essentially asLawrence, D. A., et. al. (1990) The Journal of Biological Chemistry,265, 20293-20301. Briefly, BAIT containing samples were serially dilutedin microtiter plates into 0.15 M NaCl, 0.05 M Tris-HCI, pH 7.5containing 100 μg/ml bovine serum albumin, and 0.01% Tween 80, 100 μlfinal volume. Enzyme was added (5 nM for tPA and plasmin, and 2 nM forthrombin, uPA, and trypsin), and the samples incubated for 30 minutes at23° C. Next, 100 μl of the same buffer containing 0.5 mM substrate,(Spectrozyme tPA (BioPool) for tPA, S2444 (Chromogenix) for uPA, S2390(Chromogenix) for plasmin, and chromozym TRY (Boehringer Mannheim) fortrypsin and thrombin. The plates were then were incubated at 37° C. in aThermoMax plate reader and the change in absorbance at 405 nM monitoredfor 30 minutes. The amount of inhibition was calculated from theresidual enzyme activity. These results of these assays are shown inFIG. 5 where the % inhibition of each enzyme is plotted against theconcentration of BAIT (“neural serpin”).

[0333] Thus, the invention also provides a method of treatment of anindividual in need of an increased level of BAIT activity (or,preferably, of decreased proteolytic activity of a BAIT-susceptibleprotease, particularly t-PA, trypsin, thrombin and/or urokinase-typeplasminogen activator (u-PA)) comprising administering to such anindividual a pharmaceutical composition comprising an amount of anisolated BAIT polypeptide, polynucleotide, agonist, antagonist,including antibodies, of the invention, particularly a mature form ofthe BAIT protein of the invention, effective to increase the BAITactivity level (and, preferably, thereby decreasing the BAIT-susceptibleprotease activity) in such an individual.

[0334] Additionally, the invention also provides a method of treatmentof an individual in need of a decreased level of BAIT activity (or,preferably, of increased proteolytic activity of a BAIT-susceptibleprotease, particularly t-PA, trypsin, thrombin and/or urokinase-typeplasminogen activator (u-PA)) comprising administering to such anindividual a pharmaceutical composition comprising an amount of anisolated BAIT polypeptide, polynucleotide, agonist, antagonist,including antibodies, of the invention, particularly a mature form ofthe BAIT protein of the invention, effective to decrease the BAITactivity level (and, preferably, thereby decreasing the BAIT-susceptibleprotease activity) in such an individual.

[0335] Moreover, the invention also provides a method of treatment of anindividual in need of an increase or decreased level of apoptosiscomprising administering to such an individual a pharmaceuticalcomposition comprising an amount of an isolated BAIT polypeptide,polynucleotide, agonist, antagonist, including antibodies, of theinvention, particularly a mature form of the BAIT protein of theinvention, effective to increase or decrease the BAIT activity level insuch an individual.

[0336] In one preferred embodiment, the invention provides a method oftreatment of an individual who has lacked oxygen and/or blood in thebrain (e.g., stroke, ischemia, etc.) comprising administering to such anindividual a pharmaceutical composition comprising an amount of anisolated BAIT polypeptide, polynucleotide, agonist, antagonist,including antibodies, of the invention, particularly a mature form ofthe BAIT protein of the invention, effective to treat such anindividual.

[0337] As noted above, one member in the serpin family closely relatedto BAIT is protease nexin I (PNI) or glia-derived nexin (GDN) which hasbeen shown to inhibit thrombin specifically and to promote, in vitro,neurite extension in neuroblastoma cell lines as well as primaryhippocampal, and sympathetic neurons. The PNI gene is inducedtranscriptionally and protein levels are increased following rat sciaticnerve axotomy. Other neurotrophic factors like nerve growth factor,brain-derived neurotrophic factor, and insulin-like growth factor Irespond likewise to peripheral nerve damage. Treatment of chickdeveloping motoneurons, i.e. E6-E9 lumbrosacral motoneurons whichnormally undergo apoptosis, with PNI results in increased survival ofmotoneurons. Motoneuron death experimentally induced by sciatic nervelesioning in mouse is also decreased by PNI addition. Alzheimer-diseasedbrain regions contain higher PNI/thrombin complexes compared with freePNI than do normal brains suggesting that PNI may have a role in CNSpathology.

[0338] Thus, BAIT can be used for treating peripheral neuropathies suchas ALS or multiple sclerosis. Motoneuron or sensory neuron damageresulting from spinal cord injury also may be prevented by treatmentwith BAIT. In addition, central nervous system diseases like Alzheimer'sdisease may be treated with BAIT or, preferably, a small molecule analogcapable of crossing the blood-brain barrier, which analog can beidentified according to the methods of the present invention.

[0339] Aside from the nervous system-related disorders described above,under diagnostic uses of the invention based on detecting BAITexpression, the protease inhibitory activity of BAIT protein of thepresent invention also indicates that this protein may be used fortherapeutic treatment of other conditions where excessive proteolyticactivity of a BAIT susceptible protease may be involved, particularlyt-PA. Thus, BAIT may be used to modulate the process of clot breakdown,for instance, in combination with Activase (recombinant t-PA) whichGenentech is marketing for clot dissolution after stroke. A majorproblem with the present Activase therapy is that frequently excessivehemorrhaging occurs. BAIT provides a specific inhibitor of t-PA whichwould fine tune the treatment process and not interact with other serineproteases in the nervous system. Similarly, a product called Trasylol(aprotinin), a protease inhibitor, is being marketed by Bayer forbleeding disorders. The beneficial action of this serine proteaseinhibitor in limiting blood loss after cardiopulmonary bypass has beenwidely reported.

[0340] PNI has been shown to inhibit breakdown of extracellular matrixin a fibroblast tumor cell line . Such breakdown is thought to enabletumor cells to metastasize by weakening of extracellular matrix whichnormally prevents penetration of unrelated cells through a tissue. BAITalso may be used to inhibit extracellular matrix destruction associatedwith tumors secreting a BAIT-susceptible protease, for instance, neuraltissue tumors secreting t-PA.

[0341] The BAIT composition will be formulated and dosed in a fashionconsistent with good medical practice, taking into account the clinicalcondition of the individual patient (especially the side effects oftreatment with BAIT composition alone), the site of delivery of the BAITcomposition, the method of administration, the scheduling ofadministration, and other factors known to practitioners. The “effectiveamount” of BAIT composition for purposes herein is thus determined bysuch considerations.

[0342] As a general proposition, the total pharmaceutically effectiveamount of BAIT polypeptide administered parenterally per dose will be inthe range of about 1 μg/kg/day to 10 mg/kg/day of patient body weight,although, as noted above, this will be subject to therapeuticdiscretion. More preferably, this dose is at least 0.01 mg/kg/day, andmost preferably for humans between about 0.01 and 1 mg/kg/day for thehormone. If given continuously, the BAIT polypeptide is typicallyadministered at a dose rate of about 1 μg/kg/hour to about 50μg/kg/hour, either by 1-4 injections per day or by continuoussubcutaneous infusions, for example, using a mini-pump. An intravenousbag solution may also be employed. The length of treatment needed toobserve changes and the interval following treatment for responses tooccur appears to vary depending on the desired effect.

[0343] Pharmaceutical compositions containing the BAIT of the inventionmay be administered orally, rectally, parenterally, intracistemally,intravaginally, intraperitoneally, topically (as by powders, ointments,drops or transdermal patch), bucally, or as an oral or nasal spray. By“pharmaceutically acceptable carrier” is meant a non-toxic solid,semisolid or liquid filler, diluent, encapsulating material orformulation auxiliary of any type. The term “parenteral” as used hereinrefers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrasternal, subcutaneous andintraarticular injection and infusion.

[0344] The BAIT composition is also suitably administered bysustained-release systems. Suitable examples of sustained-releasecompositions include semi-permeable polymer matrices in the form ofshaped articles, e.g., films, or microcapsules. Sustained-releasematrices include polylactides (U.S. Pat. No. 3,773,919, EP 58,481),copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman, U. etal., Biopolymers 22:547-556 (1983)), poly (2-hydroxyethyl methacrylate)(R. Langer et al., J. Biomed. Mater. Res. 15:167-277 (198 1), and R.Langer, Chem. Tech. 12:98-105 (1982)), ethylene vinyl acetate (R. Langeret al., Id.) or poly-D-(−)-3-hydroxybutyric acid (EP 133,988).Sustained-release BAIT compositions also include liposomally entrappedBAIT polypeptide. Lipsomes containing BAIT are prepared by methods knownper se: DE 3,218,12 1; Epstein et al., Proc. Natl. Acad. Sci. (USA)82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. (USA)77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and4,544,545; and EP 102,324. Ordinarily, the liposomes are of the small(about 200-800 Angstroms) unilamellar type in which the lipid content isgreater than about 30 mol. percent cholesterol, the selected proportionbeing adjusted for the optimal BAIT polypeptide therapy.

[0345] For parenteral administration, in one embodiment, the BAIT isformulated generally by mixing it at the desired degree of purity, in aunit dosage injectable form (solution, suspension, or emulsion), with apharmaceutically acceptable carrier, i.e., one that is non-toxic torecipients at the dosages and concentrations employed and is compatiblewith other ingredients of the formulation. For example, the formulationpreferably does not include oxidizing agents and other compounds thatare known to be deleterious to polypeptides.

[0346] Generally, the formulations are prepared by contacting the BAITcomposition (and, optionally, any cofactor which may enhance itsactivity) uniformly and intimately with liquid carriers or finelydivided solid carriers or both. Then, if necessary, the product, isshaped into the desired formulation. Preferably the carrier is aparenteral carrier, more preferably a solution that is isotonic with theblood of the recipient. Examples of such carrier vehicles include water,saline, Ringer's solution, and dextrose solution. Non-aqueous vehiclessuch as fixed oils and ethyl oleate are also useful herein, as well asliposomes.

[0347] The carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, manose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium; and/or nonionicsurfactants such as polysorbates, poloxamers, or PEG.

[0348] The BAIT polypeptide is typically formulated in such vehicles ata concentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml,at a pH of about 3 to 8. It will be understood that the use of certainof the foregoing excipients, carriers, or stabilizers will result in theformation of BAIT polypeptide salts.

[0349] BAIT composition to be used for therapeutic administration mustbe sterile. Sterility is readily accomplished by filtration throughsterile filtration membranes (e.g., 0.2 micron membranes). TherapeuticBAIT compositions generally are placed into a container having a sterileaccess port, for example, an intravenous solution bag or vial having astopper pierceable by a hypodermic injection needle.

[0350] BAIT composition ordinarily will be stored in unit or multi-dosecontainers, for example, sealed ampoules or vials, as an aqueoussolution or as a lyophilized formulation for reconstitution. As anexample of a lyophilized formulation, 10-ml vials are filled with 5 mlof sterile-filtered 1% (w/v) aqueous BAIT polypeptide solution, and theresulting mixture is lyophilized. The infusion solution is prepared byreconstituting the lyophilized BAIT polypeptide using bacteriostaticWater-for- Injection.

[0351] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention.Associated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the acency of manufacture, use or sale for human administration. Inaddition, the polypeptides of the present invention may be employed inconjunction with other therapeutic compounds.

[0352] Agonists and Antagonists—Assays and Molecules

[0353] The invention also provides a method of screening compounds toidentify those which enhance or block the action of BAIT on proteases,such as its interaction with proteases or with protein cofactors such asextracellular matrix proteins. Thus, protease-inhibiting activity ofanother serpin, plasminogen activator inhibitor-1 (PAI-1), is known tobe modulated by its protein cofactor, vitronectin, which binds to activePAL-1 and prevents its spontaneous conversion to a latent form. See, forinstance, Reilly, T. M., et al., supra. Similarly, heparin is known toenhance the activity of antithrombin III and several other serpins. Thepresent invention provides an assay for identifying such a protein orother cofactor which binds to BAIT and thereby modulates itsanti-proteolytic activity. In general, therefore, an agonist in thepresent context is a compound which increases the natural biologicalfunctions of BAIT or which functions in a manner similar to BAIT, whileantagonists decrease or eliminate such functions.

[0354] For example, a cellular compartment, such as a membrane or apreparation thereof, such as a membrane-preparation, may be preparedfrom a cell that expresses a molecule that binds BAIT, such as amolecule of a signaling or regulatory pathway modulated by BAIT. Thepreparation is incubated with labeled BAIT in the absence or thepresence of a candidate molecule which may be a BAIT agonist orantagonist. The ability of the candidate molecule to bind the bindingmolecule is reflected in decreased binding of the labeled ligand.Molecules which bind gratuitously, i.e., without inducing the effects ofBAIT on binding the BAIT binding molecule, are most likely to be goodantagonists. Molecules that bind well and elicit effects that are thesame as or closely related to BAIT are agonists.

[0355] BAIT-like effects of potential agonists and antagonists may bemeasured, for instance, by determining activity of a second messengersystem following interaction of the candidate molecule with a cell orappropriate cell preparation, and comparing the effect with that of BAITor molecules that elicit the same effects as BAIT. Second messengersystems that may be useful in this regard include but are not limited toAMP guanylate cyclase, ion channel or phosphoinositide hydrolysis secondmessenger systems.

[0356] Another example of an assay for BAIT antagonists is a competitiveassay that combines BAIT and a potential antagonist BAIT-susceptibleprotease, particularly t-PA, under appropriate conditions for acompetitive inhibition assay. BAIT can be labeled, such as byradioactivity, such that the number of BAIT molecules bound to proteasemolecules can be determined accurately to assess the effectiveness ofthe potential antagonist.

[0357] Potential antagonists include small organic molecules, peptides,polypeptides and antibodies that bind to a polypeptide of the inventionand thereby inhibit or extinguish its activity. Potential antagonistsalso may be small organic molecules, a peptide, a polypeptide such as aclosely related protein or antibody that binds the same sites on abinding molecule, such as BAIT susceptible protease molecule, withoutinducing BAIT-induced activities, thereby preventing the action of BAITby excluding BAIT from binding.

[0358] Other potential antagonists include antisense molecules.Antisense technology can be used to control gene expression throughantisense DNA or RNA or through triple-helix formation. Antisensetechniques are discussed, for example, in Okano, J. Neurochem. 56: 560(1991); “Oligodeoxynucleotides as Antisense Inhibitors of GeneExpression,” CRC Press, Boca Raton, Fla. (1988). Triple helix formationis discussed in, for instance Lee et al., Nucleic Acids Research 6: 3073(1979); Cooney et al., Science 241: 456 (1988); and Dervan et al.,Science 251: 1360 (1991). The methods are based on binding of apolynucleotide to a complementary DNA or RNA. For example, the 5′ codingportion of a polynucleotide that encodes the mature polypeptide of thepresent invention may be used to design an antisense RNA oligonucleotideof from about 10 to 40 base pairs in length. A DNA oligonucleotide isdesigned to be complementary to a region of the gene involved intranscription thereby preventing transcription and the production ofBAIT. The antisense RNA oligonucleotide hybridizes to the mRNA in vivoand blocks translation of the mRNA molecule into BAIT polypeptide. Theoligonucleotides described above can also be delivered to cells suchthat the antisense RNA or DNA may be expressed in vivo to inhibitproduction of BAIT.

[0359] The agonists and antagonists may be employed in a compositionwith a pharmaceutically acceptable carrier, e.g., as described above.

[0360] The BAIT agonists may be employed in place of a BAIT polypeptide,for instance, for treating peripheral neuropathies such as ALS ormultiple sclerosis. Motoneuron or sensory neuron damage resulting fromspinal cord injury also may be prevented by treatment with BAITagonists. In addition, central nervous system diseases like Alzheimer'sdisease may be treated a small molecule agonist capable of crossing theblood-brain barrier, which analog can be identified according to themethods of the present invention. BAIT agonists also may be used fortherapeutic treatment of other conditions where excessive proteolyticactivity of a BAIT susceptible protease may be involved, particularlyt-PA. Thus, BAIT may be used to modulate the process of clot breakdown,for instance, in combination with Activase (recombinant t-PA) for clotdissolution after stroke. Further, BAIT agonists also may be used toinhibit extracellular matrix destruction associated with tumorssecreting a BAIT-susceptible protease, for instance, neural tissuetumors secreting t-PA.

[0361] The BAIT antagonists may be used in a method for treating anindividual in need of a decreased level of BAIT activity in the body(i.e., less inhibition of a protease susceptible to BAIT) comprisingadministering to such an individual a composition comprising atherapeutically effective amount of a BAIT antagonist. As noted above,elimination of a serpin inhibitor of u-PA, PNI (described above) byhomologous recombination leads to reduced long-term potentiation (LTP)of learning, whereas overexpression of PNI results in enhanced LTP ofhippocampal neurons. Id. Similarly, antagonists of BAIT activity capableof passing the blood-brain barrier, by mimicking overexpression of BAIT,can be used to enhance LTP of hippocampal neurons in nervous systemconditions characterized by excessive BAIT expression.

[0362] Neurological Diseases

[0363] Nervous system diseases, disorders, and/or conditions, which canbe treated, prevented, and/or diagnosed with the compositions of theinvention (e.g., polypeptides, polynucleotides, and/or agonists orantagonists, including antibodies), include, but are not limited to,nervous system injuries, and diseases, disorders, and/or conditionswhich result in either a disconnection of axons, a diminution ordegeneration of neurons, or demyelination. Nervous system lesions whichmay be treated, prevented, and/or diagnosed in a patient (includinghuman and non-human mammalian patients) according to the invention,include but are not limited to, the following lesions of either thecentral (including spinal cord, brain) or peripheral nervous systems:(1) ischemic lesions, in which a lack of oxygen in a portion of thenervous system results in neuronal injury or death, including cerebralinfarction or ischemia, or spinal cord infarction or ischemia; (2)traumatic lesions, including lesions caused by physical injury orassociated with surgery, for example, lesions which sever a portion ofthe nervous system, or compression injuries; (3) malignant lesions, inwhich a portion of the nervous system is destroyed or injured bymalignant tissue which is either a nervous system associated malignancyor a malignancy derived from non-nervous system tissue; (4) infectiouslesions, in which a portion of the nervous system is destroyed orinjured as a result of infection, for example, by an abscess orassociated with infection by human immunodeficiency virus, herpeszoster, or herpes simplex virus or with Lyme disease, tuberculosis,syphilis; (5) degenerative lesions, in which a portion of the nervoussystem is destroyed or injured as a result of a degenerative processincluding but not limited to degeneration associated with Parkinson'sdisease, Alzheimer's disease, Huntington's chorea, or amyotrophiclateral sclerosis (ALS); (6) lesions associated with nutritionaldiseases, disorders, and/or conditions, in which a portion of thenervous system is destroyed or injured by a nutritional disorder ordisorder of metabolism including but not limited to, vitamin B12deficiency, folic acid deficiency, Wemicke disease, tobacco-alcoholamblyopia, Marchiafava-Bignami disease (primary degeneration of thecorpus callosum), and alcoholic cerebellar degeneration; (7)neurological lesions associated with systemic diseases including, butnot limited to, diabetes (diabetic neuropathy, Bell's palsy), systemiclupus erythematosus, carcinoma, or sarcoidosis; (8) lesions caused bytoxic substances including alcohol, lead, or particular neurotoxins; and(9) demyelinated lesions in which a portion of the nervous system isdestroyed or injured by a demyelinating disease including, but notlimited to, multiple sclerosis, human immunodeficiency virus-associatedmyelopathy, transverse myelopathy or various etiologies, progressivemultifocal leukoencephalopathy, and central pontine myelinolysis.

[0364] In a preferred embodiment, the polypeptides, polynucleotides, oragonists or antagonists of the invention are used to protect neuralcells from the damaging effects of cerebral hypoxia. According to thisembodiment, the compositions of the invention are used to treat,prevent, and/or diagnose neural cell injury associated with cerebralhypoxia. In one aspect of this embodiment, the polypeptides,polynucleotides, or agonists or antagonists of the invention are used totreat, prevent, and/or diagnose neural cell injury associated withcerebral ischemia. In another aspect of this embodiment, thepolypeptides, polynucleotides, or agonists or antagonists of theinvention are used to treat, prevent, and/or diagnose neural cell injuryassociated with cerebral infarction. In another aspect of thisembodiment, the polypeptides, polynucleotides, or agonists orantagonists of the invention are used to treat, prevent, and/or diagnoseor prevent neural cell injury associated with a stroke. In a furtheraspect of this embodiment, the polypeptides, polynucleotides, oragonists or antagonists of the invention are used to treat, prevent,and/or diagnose neural cell injury associated with a heart attack.

[0365] The compositions of the invention which are useful for treatingor preventing a nervous system disorder may be selected by testing forbiological activity in promoting the survival or differentiation ofneurons. For example, and not by way of limitation, compositions of theinvention which elicit any of the following effects may be usefulaccording to the invention: (1) increased survival time of neurons inculture; (2) increased sprouting of neurons in culture or in vivo; (3)increased production of a neuron-associated molecule in culture or invivo, e.g., choline acetyltransferase or acetylcholinesterase withrespect to motor neurons; or (4) decreased symptoms of neurondysfunction in vivo. Such effects may be measured by any method known inthe art. In preferred, non-limiting embodiments, increased survival ofneurons may routinely be measured using a method set forth herein orotherwise known in the art, such as, for example, the method set forthin Arakawa et al. (J. Neurosci. 10:3507-3515 (1990)); increasedsprouting of neurons may be detected by methods known in the art, suchas, for example, the methods set forth in Pestronk et al. (Exp. Neurol.70:65-82 (1980)) or Brown et al. (Ann. Rev. Neurosci. 4:17-42 (1981));increased production of neuron-associated molecules may be measured bybioassay, enzymatic assay, antibody binding, Northern blot assay, etc.,using techniques known in the art and depending on the molecule to bemeasured; and motor neuron dysfunction may be measured by assessing thephysical manifestation of motor neuron disorder, e.g., weakness, motorneuron conduction velocity, or functional disability.

[0366] In specific embodiments, motor neuron diseases, disorders, and/orconditions that may be treated, prevented, and/or diagnosed according tothe invention include, but are not limited to, diseases, disorders,and/or conditions such as infarction, infection, exposure to toxin,trauma, surgical damage, degenerative disease or malignancy that mayaffect motor neurons as well as other components of the nervous system,as well as diseases, disorders, and/or conditions that selectivelyaffect neurons such as amyotrophic lateral sclerosis, and including, butnot limited to, progressive spinal muscular atrophy, progressive bulbarpalsy, primary lateral sclerosis, infantile and juvenile muscularatrophy, progressive bulbar paralysis of childhood (Fazio-Londesyndrome), poliomyelitis and the post polio syndrome, and HereditaryMotorsensory Neuropathy (Charcot-Marie-Tooth Disease).

[0367] Binding Activity

[0368] A polypeptide of the present invention may be used to screen formolecules that bind to the polypeptide or for molecules to which thepolypeptide binds. The binding of the polypeptide and the molecule mayactivate (agonist), increase, inhibit (antagonist), or decrease activityof the polypeptide or the molecule bound. Examples of such moleculesinclude antibodies, oligonucleotides, proteins (e.g., receptors),orsmall molecules.

[0369] Preferably, the molecule is closely related to the natural ligandof the polypeptide, e.g., a fragment of the ligand, or a naturalsubstrate, a ligand, a structural or functional mimetic. (See, Coliganet al., Current Protocols in Immunology 1(2):Chapter 5 (1991).)Similarly, the molecule can be closely related to the natural receptorto which the polypeptide binds, or at least, a fragment of the receptorcapable of being bound by the polypeptide (e.g., active site). In eithercase, the molecule can be rationally designed using known techniques.

[0370] Preferably, the screening for these molecules involves producingappropriate cells which express the polypeptide, either as a secretedprotein or on the cell membrane. Preferred cells include cells frommammals, yeast, Drosophila, or E. coli. Cells expressing the polypeptide(or cell membrane containing the expressed polypeptide) are thenpreferably contacted with a test compound potentially containing themolecule to observe binding, stimulation, or inhibition of activity ofeither the polypeptide or the molecule.

[0371] The assay may simply test binding of a candidate compound to thepolypeptide, wherein binding is detected by a label, or in an assayinvolving competition with a labeled competitor. Further, the assay maytest whether the candidate compound results in a signal generated bybinding to the polypeptide.

[0372] Alternatively, the assay can be carried out using cell-freepreparations, polypeptide/molecule affixed to a solid support, chemicallibraries, or natural product mixtures. The assay may also simplycomprise the steps of mixing a candidate compound with a solutioncontaining a polypeptide, measuring polypeptide/molecule activity orbinding, and comparing the polypeptide/molecule activity or binding to astandard.

[0373] Preferably, an ELISA assay can measure polypeptide level oractivity in a sample (e.g., biological sample) using a monoclonal orpolyclonal antibody. The antibody can measure polypeptide level oractivity by either binding, directly or indirectly, to the polypeptideor by competing with the polypeptide for a substrate.

[0374] Additionally, the receptor to which a polypeptide of theinvention binds can be identified by numerous methods known to those ofskill in the art, for example, ligand panning and FACS sorting (Coligan,et al., Current Protocols in Immun., 1(2), Chapter 5, (1991)). Forexample, expression cloning is employed wherein polyadenylated RNA isprepared from a cell responsive to the polypeptides, for example, NIH3T3cells which are known to contain multiple receptors for the FGF familyproteins, and SC-3 cells, and a cDNA library created from this RNA isdivided into pools and used to transfect COS cells or other cells thatare not responsive to the polypeptides. Transfected cells which aregrown on glass slides are exposed to the polypeptide of the presentinvention, after they have been labelled. The polypeptides can belabeled by a variety of means including iodination or inclusion of arecognition site for a site-specific protein kinase.

[0375] Following fixation and incubation, the slides are subjected toauto-radiographic analysis. Positive pools are identified and sub-poolsare prepared and re-transfected using an iterative sub-pooling andre-screening process, eventually yielding a single clones that encodesthe putative receptor.

[0376] As an alternative approach for receptor identification, thelabeled polypeptides can be photoaffinity linked with cell membrane orextract preparations that express the receptor molecule. Cross-linkedmaterial is resolved by PAGE analysis and exposed to X-ray film. Thelabeled complex containing the receptors of the polypeptides can beexcised, resolved into peptide fragments, and subjected to proteinmicrosequencing. The amino acid sequence obtained from microsequencingwould be used to design a set of degenerate oligonucleotide probes toscreen a cDNA library to identify the genes encoding the putativereceptors.

[0377] Moreover, the techniques of gene-shuffling, motif-shuffling,exon-shuffling, and/or codon-shuffling (collectively referred to as “DNAshuffling”) may be employed to modulate the activities of polypeptidesof the invention thereby effectively generating agonists and antagonistsof polypeptides of the invention. See generally, U.S. Pat. Nos.5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458, and Patten,P. A., et al., Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama, S.Trends Biotechnol. 16(2):76-82 (1998); Hansson, L. O., et al., J. Mol.Biol. 287:265-76 (1999); and Lorenzo, M. M. and Blasco, R. Biotechniques24(2):308-13 (1998) (each of these patents and publications are herebyincorporated by reference). In one embodiment, alteration ofpolynucleotides and corresponding polypeptides of the invention may beachieved by DNA shuffling. DNA shuffling involves the assembly of two ormore DNA segments into a desired polynucleotide sequence of theinvention molecule by homologous, or site-specific, recombination. Inanother embodiment, polynucleotides and corresponding polypeptides ofthe invention may be alterred by being subjected to random mutagenesisby error-prone PCR, random nucleotide insertion or other methods priorto recombination. In another embodiment, one or more components, motifs,sections, parts, domains, fragments, etc., of the polypeptides of theinvention may be recombined with one or more components, motifs,sections, parts, domains, fragments, etc. of one or more heterologousmolecules. In preferred embodiments, the heterologous molecules arefamily members. In further preferred embodiments, the heterologousmolecule is a growth factor such as, for example, platelet-derivedgrowth factor (PDGF), insulin-like growth factor (IGF-I), transforminggrowth factor (TGF)-alpha, epidermal growth factor (EGF), fibroblastgrowth factor (FGF), TGF-beta, bone morphogenetic protein (BMP)-2,BMP-4, BMP-5, BMP-6, BMP-7, activins A and B, decapentaplegic(dpp), 60A,OP-2, dorsalin, growth differentiation factors (GDFs), nodal, MIS,inhibin-alpha, TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta5, andglial-derived neurotrophic factor (GDNF).

[0378] Other preferred fragments are biologically active fragments ofthe polypeptides of the invention. Biologically active fragments arethose exhibiting activity similar, but not necessarily identical, to anactivity of the polypeptide. The biological activity of the fragmentsmay include an improved desired activity, or a decreased undesirableactivity.

[0379] Additionally, this invention provides a method of screeningcompounds to identify those which modulate the action of the polypeptideof the present invention. An example of such an assay comprisescombining a mammalian fibroblast cell, a the polypeptide of the presentinvention, the compound to be screened and 3[H] thymidine under cellculture conditions where the fibroblast cell would normally proliferate.A control assay may be performed in the absence of the compound to bescreened and compared to the amount of fibroblast proliferation in thepresence of the compound to determine if the compound stimulatesproliferation by determining the uptake of 3[H] thymidine in each case.The amount of fibroblast cell proliferation is measured by liquidscintillation chromatography which measures the incorporation of 3[H]thymidine. Both agonist and antagonist compounds may be identified bythis procedure.

[0380] In another method, a mammalian cell or membrane preparationexpressing a receptor for a polypeptide of the present invention isincubated with a labeled polypeptide of the present invention in thepresence of the compound. The ability of the compound to enhance orblock this interaction could then be measured. Alternatively, theresponse of a known second messenger system following interaction of acompound to be screened and the receptor is measured and the ability ofthe compound to bind to the receptor and elicit a second messengerresponse is measured to determine if the compound is a potential agonistor antagonist. Such second messenger systems include but are not limitedto, cAMP guanylate cyclase, ion channels or phosphoinositide hydrolysis.

[0381] All of these above assays can be used as diagnostic or prognosticmarkers. The molecules discovered using these assays can be used totreat, prevent, and/or diagnose disease or to bring about a particularresult in a patient (e.g., blood vessel growth) by activating orinhibiting the polypeptide/molecule. Moreover, the assays can discoveragents which may inhibit or enhance the production of the polypeptidesof the invention from suitably manipulated cells or tissues. Therefore,the invention includes a method of identifying compounds which bind tothe polypeptides of the invention comprising the steps of: (a)incubating a candidate binding compound with the polypeptide; and (b)determining if binding has occurred. Moreover, the invention includes amethod of identifying agonists/antagonists comprising the steps of: (a)incubating a candidate compound with the polypeptide, (b) assaying abiological activity, and (b) determining if a biological activity of thepolypeptide has been altered.

[0382] Also, one could identify molecules bind a polypeptide of theinvention experimentally by using the beta-pleated sheet regionscontained in the polypeptide sequence of the protein. Accordingly,specific embodiments of the invention are directed to polynucleotidesencoding polypeptides which comprise, or alternatively consist of, theamino acid sequence of each beta pleated sheet regions in a disclosedpolypeptide sequence. Additional embodiments of the invention aredirected to polynucleotides encoding polypeptides which comprise, oralternatively consist of, any combination or all of contained in thepolypeptide sequences of the invention. Additional preferred embodimentsof the invention are directed to polypeptides which comprise, oralternatively consist of, the amino acid sequence of each of the betapleated sheet regions in one of the polypeptide sequences of theinvention. Additional embodiments of the invention are directed topolypeptides which comprise, or alternatively consist of, anycombination or all of the beta pleated sheet regions in one of thepolypeptide sequences of the invention.

[0383] Targeted Delivery In another embodiment, the invention provides amethod of delivering compositions to targeted cells expressing areceptor for a polypeptide of the invention, or cells expressing a cellbound form of a polypeptide of the invention.

[0384] As discussed herein, polypeptides or antibodies of the inventionmay be associated with heterologous polypeptides, heterologous nucleicacids, toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/orcovalent interactions. In one embodiment, the invention provides amethod for the specific delivery of compositions of the invention tocells by administering polypeptides of the invention (includingantibodies) that are associated with heterologous polypeptides ornucleic acids. In one example, the invention provides a method fordelivering a therapeutic protein into the targeted cell. In anotherexample, the invention provides a method for delivering a singlestranded nucleic acid (e.g., antisense or ribozymes) or double strandednucleic acid (e.g., DNA that can integrate into the cell's genome orreplicate episomally and that can be transcribed) into the targetedcell.

[0385] In another embodiment, the invention provides a method for thespecific destruction of cells (e.g., the destruction of tumor cells) byadministering polypeptides of the invention (e.g., polypeptides of theinvention or antibodies of the invention) in association with toxins orcytotoxic prodrugs.

[0386] By “toxin” is meant compounds that bind and activate endogenouscytotoxic effector systems, radioisotopes, holotoxins, modified toxins,catalytic subunits of toxins, or any molecules or enzymes not normallypresent in or on the surface of a cell that under defined conditionscause the cell's death. Toxins that may be used according to the methodsof the invention include, but are not limited to, radioisotopes known inthe art, compounds such as, for example, antibodies (or complementfixing containing portions thereof) that bind an inherent or inducedendogenous cytotoxic effector system, thymidine kinase, endonuclease,RNAse, alpha toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheriatoxin, saporin, momordin, gelonin, pokeweed antiviral protein,alpha-sarcin and cholera toxin. By “cytotoxic prodrug” is meant anon-toxic compound that is converted by an enzyme, normally present inthe cell, into a cytotoxic compound. Cytotoxic prodrugs that may be usedaccording to the methods of the invention include, but are not limitedto, glutamyl derivatives of benzoic acid mustard alkylating agent,phosphate derivatives of etoposide or mitomycin C, cytosine arabinoside,daunorubisin, and phenoxyacetamide derivatives of doxorubicin.

[0387] Drug Screening

[0388] Further contemplated is the use of the polypeptides of thepresent invention, or the polynucleotides encoding these polypeptides,to screen for molecules which modify the activities of the polypeptidesof the present invention. Such a method would include contacting thepolypeptide of the present invention with a selected compound(s)suspected of having antagonist or agonist activity, and assaying theactivity of these polypeptides following binding.

[0389] This invention is particularly useful for screening therapeuticcompounds by using the polypeptides of the present invention, or bindingfragments thereof, in any of a variety of drug screening techniques. Thepolypeptide or fragment employed in such a test may be affixed to asolid support, expressed on a cell surface, free in solution, or locatedintracellularly. One method of drug screening utilizes eukaryotic orprokaryotic host cells which are stably transformed with recombinantnucleic acids expressing the polypeptide or fragment. Drugs are screenedagainst such transformed cells in competitive binding assays. One maymeasure, for example, the formulation of complexes between the agentbeing tested and a polypeptide of the present invention.

[0390] Thus, the present invention provides methods of screening fordrugs or any other agents which affect activities mediated by thepolypeptides of the present invention. These methods comprise contactingsuch an agent with a polypeptide of the present invention or a fragmentthereof and assaying for the presence of a complex between the agent andthe polypeptide or a fragment thereof, by methods well known in the art.In such a competitive binding assay, the agents to screen are typicallylabeled. Following incubation, free agent is separated from that presentin bound form, and the amount of free or uncomplexed label is a measureof the ability of a particular agent to bind to the polypeptides of thepresent invention.

[0391] Another technique for drug screening provides high throughputscreening for compounds having suitable binding affinity to thepolypeptides of the present invention, and is described in great detailin European Patent Application 84/03564, published on Sep. 13, 1984,which is incorporated herein by reference herein. Briefly stated, largenumbers of different small peptide test compounds are synthesized on asolid substrate, such as plastic pins or some other surface. The peptidetest compounds are reacted with polypeptides of the present inventionand washed. Bound polypeptides are then detected by methods well knownin the art. Purified polypeptides are coated directly onto plates foruse in the aforementioned drug screening techniques. In addition,non-neutralizing antibodies may be used to capture the peptide andimmobilize it on the solid support.

[0392] This invention also contemplates the use of competitive drugscreening assays in which neutralizing antibodies capable of bindingpolypeptides of the present invention specifically compete with a testcompound for binding to the polypeptides or fragments thereof. In thismanner, the antibodies are used to detect the presence of any peptidewhich shares one or more antigenic epitopes with a polypeptide of theinvention.

[0393] Antisense and Ribozyme (Antagonists)

[0394] In specific embodiments, antagonists according to the presentinvention are nucleic acids corresponding to the sequences contained inSEQ ID NO:1, or the complementary strand thereof, and/or to nucleotidesequences contained a deposited clone. In one embodiment, antisensesequence is generated internally by the organism, in another embodiment,the antisense sequence is separately administered (see, for example,O'Connor, Neurochem., 56:560 (1991). Oligodeoxynucleotides as AnitsenseInhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988).Antisense technology can be used to control gene expression throughantisense DNA or RNA, or through triple-helix formation. Antisensetechniques are discussed for example, in Okano, Neurochem., 56:560(1991); Oligodeoxynucleotides as Antisense Inhibitors of GeneExpression, CRC Press, Boca Raton, Fla. (1988). Triple helix formationis discussed in, for instance, Lee et al., Nucleic Acids Research,6:3073 (1979); Cooney et al., Science, 241:456 (1988); and Dervan etal., Science, 251:1300 (1991). The methods are based on binding of apolynucleotide to a complementary DNA or RNA.

[0395] For example, the use of c-myc and c-myb antisense RNA constructsto inhibit the growth of the non-lymphocytic leukemia cell line HL-60and other cell lines was previously described. (Wickstrom et al. (1988);Anfossi et al. (1989)). These experiments were performed in vitro byincubating cells with the oligoribonucleotide. A similar procedure forin vivo use is described in WO 91/15580. Briefly, a pair ofoligonucleotides for a given antisense RNA is produced as follows: Asequence complimentary to the first 15 bases of the open reading frameis flanked by an EcoR1 site on the 5 end and a HindIII site on the 3end. Next, the pair of oligonucleotides is heated at 90° C. for oneminute and then annealed in 2× ligation buffer (20 mM TRIS HCl pH 7.5,10 mM MgCl2, 10 MM dithiothreitol (DTT) and 0.2 mM ATP) and then ligatedto the EcoR1/Hind III site of the retroviral vector PMV7 (WO 91/15580).

[0396] For example, the 5′ coding portion of a polynucleotide thatencodes the mature polypeptide of the present invention may be used todesign an antisense RNA oligonucleotide of from about 10 to 40 basepairs in length. A DNA oligonucleotide is designed to be complementaryto a region of the gene involved in transcription thereby preventingtranscription and the production of the receptor. The antisense RNAoligonucleotide hybridizes to the mRNA in vivo and blocks translation ofthe mRNA molecule into receptor polypeptide.

[0397] In one embodiment, the antisense nucleic acid of the invention isproduced intracellularly by transcription from an exogenous sequence.For example, a vector or a portion thereof, is transcribed, producing anantisense nucleic acid (RNA) of the invention. Such a vector wouldcontain a sequence encoding the antisense nucleic acid of the invention.Such a vector can remain episomal or become chromosomally integrated, aslong as it can be transcribed to produce the desired antisense RNA. Suchvectors can be constructed by recombinant DNA technology methodsstandard in the art. Vectors can be plasmid, viral, or others known inthe art, used for replication and expression in vertebrate cells.Expression of the sequence encoding a polypeptide of the invention, orfragments thereof, can be by any promoter known in the art to act invertebrate, preferably human cells. Such promoters can be inducible orconstitutive. Such promoters include, but are not limited to, the SV40early promoter region (Bernoist and Chambon, Nature, 29:304-310 (1981),the promoter contained in the 3′ long terminal repeat of Rous sarcomavirus (Yamamoto et al., Cell, 22:787-797 (1980), the herpes thymidinepromoter (Wagner et al., Proc. Natl. Acad. Sci. U.S.A., 78:1441-1445(1981), the regulatory sequences of the metallothionein gene (Brinsteret al., Nature, 296:39-42 (1982)), etc.

[0398] The antisense nucleic acids of the invention comprise a sequencecomplementary to at least a portion of an RNA transcript of a gene ofinterest. However, absolute complementarity, although preferred, is notrequired. A sequence “complementary to at least a portion of an RNA,”referred to herein, means a sequence having sufficient complementarityto be able to hybridize with the RNA, forming a stable duplex; in thecase of double stranded antisense nucleic acids of the invention, asingle strand of the duplex DNA may thus be tested, or triplex formationmay be assayed. The ability to hybridize will depend on both the degreeof complementarity and the length of the antisense nucleic acidGenerally, the larger the hybridizing nucleic acid, the more basemismatches with a RNA sequence of the invention it may contain and stillform a stable duplex (or triplex as the case may be). One skilled in theart can ascertain a tolerable degree of mismatch by use of standardprocedures to determine the melting point of the hybridized complex.

[0399] Oligonucleotides that are complementary to the 5′ end of themessage, e.g., the 5′ untranslated sequence up to and including the AUGinitiation codon, should work most efficiently at inhibitingtranslation. However, sequences complementary to the 3′ untranslatedsequences of mRNAs have been shown to be effective at inhibitingtranslation of mRNAs as well. See generally, Wagner, R., Nature,372:333-335 (1994). Thus, oligonucleotides complementary to either the5′- or 3′-non-translated, non-coding regions of a polynucleotidesequence of the invention could be used in an antisense approach toinhibit translation of endogenous mRNA. Oligonucleotides complementaryto the 5′ untranslated region of the mRNA should include the complementof the AUG start codon. Antisense oligonucleotides complementary to mRNAcoding regions are less efficient inhibitors of translation but could beused in accordance with the invention. Whether designed to hybridize tothe 5′-, 3′- or coding region of mRNA, antisense nucleic acids should beat least six nucleotides in length, and are preferably oligonucleotidesranging from 6 to about 50 nucleotides in length. In specific aspectsthe oligonucleotide is at least 10 nucleotides, at least 17 nucleotides,at least 25 nucleotides or at least 50 nucleotides.

[0400] The polynucleotides of the invention can be DNA or RNA orchimeric mixtures or derivatives or modified versions thereof,single-stranded or double- stranded. The oligonucleotide can be modifiedat the base moiety, sugar moiety, or phosphate backbone, for example, toimprove stability of the molecule, hybridization, etc. Theoligonucleotide may include other appended groups such as peptides(e.g., for targeting host cell receptors in vivo), or agentsfacilitating transport across the cell membrane (see, e.g., Letsinger etal., Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556 (1989); Lemaitre et al.,Proc. Natl. Acad. Sci., 84:648-652 (1987); PCT Publication NO:WO88/09810, published Dec. 15, 1988) or the blood-brain barrier (see,e.g., PCT Publication NO: WO89/10134, published Apr. 25, 1988),hybridization-triggered cleavage agents. (See, e.g., Krol et al.,BioTechniques, 6:958-976 (1988)) or intercalating agents. (See, e.g.,Zon, Pharm. Res., 5:539-549 (1988)). To this end, the oligonucleotidemay be conjugated to another molecule, e.g., a peptide, hybridizationtriggered cross-linking agent, transport agent, hybridization-triggeredcleavage agent, etc.

[0401] The antisense oligonucleotide may comprise at least one modifiedbase moiety which is selected from the group including, but not limitedto, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, b eta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-metho xyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

[0402] The antisense oligonucleotide may also comprise at least onemodified sugar moiety selected from the group including, but not limitedto, arabinose, 2-fluoroarabinose, xylulose, and hexose.

[0403] In yet another embodiment, the antisense oligonucleotidecomprises at least one modified phosphate backbone selected from thegroup including, but not limited to, a phosphorothioate, aphosphorodithioate, a phosphoramidothioate, a phosphoramidate, aphosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and aformacetal or analog thereof.

[0404] In yet another embodiment, the antisense oligonucleotide is ana-anomeric ligonucleotide. An a-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual b-units, the strands run parallel to each other (Gautier et al.,Nucl. Acids Res., 15:6625-6641 (1987)). The oligonucleotide is a2-0-methylribonucleotide (Inoue et al., Nucl. Acids Res., 15:6131-6148(1987)), or a chimeric RNA-DNA analogue (Inoue et al., FEBS Lett.215:327-330 (1987)).

[0405] Polynucleotides of the invention may be synthesized by standardmethods known in the art, e.g. by use of an automated DNA synthesizer(such as are commercially available from Biosearch, Applied Biosystems,etc.). As examples, phosphorothioate oligonucleotides may be synthesizedby the method of Stein et al. (Nucl. Acids Res., 16:3209 (1988)),methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., Proc. Natl. Acad. Sci.U.S.A., 85:7448-7451 (1988)), etc.

[0406] While antisense nucleotides complementary to the coding regionsequence of the invention could be used, those complementary to thetranscribed untranslated region are most preferred.

[0407] Potential antagonists according to the invention also includecatalytic RNA, or a ribozyme (See, e.g., PCT International PublicationWO 90/11364, published October 4, 1990; Sarver et al, Science,247:1222-1225 (1990). While ribozymes that cleave mRNA at site specificrecognition sequences can be used to destroy mRNAs corresponding to thepolynucleotides of the invention, the use of hammerhead ribozymes ispreferred. Hammerhead ribozymes cleave mRNAs at locations dictated byflanking regions that form complementary base pairs with the targetmRNA. The sole requirement is that the target mRNA have the followingsequence of two bases: 5′-UG-3′. The construction and production ofhammerhead ribozymes is well known in the art and is described morefully in Haseloff and Gerlach, Nature, 334:585-591 (1988). There arenumerous potential hammerhead ribozyme cleavage sites within eachnucleotide sequence disclosed in the sequence listing. Preferably, theribozyme is engineered so that the cleavage recognition site is locatednear the 5′ end of the mRNA corresponding to the polynucleotides of theinvention; i.e., to increase efficiency and minimize the intracellularaccumulation of non-functional mRNA transcripts.

[0408] As in the antisense approach, the ribozymes of the invention canbe composed of modified oligonucleotides (e.g. for improved stability,targeting, etc.) and should be delivered to cells which express thepolynucleotides of the invention in vivo. DNA constructs encoding theribozyme may be introduced into the cell in the same manner as describedabove for the introduction of antisense encoding DNA. A preferred methodof delivery involves using a DNA construct “encoding” the ribozyme underthe control of a strong constitutive promoter, such as, for example, polIII or pol II promoter, so that transfected cells will producesufficient quantities of the ribozyme to destroy endogenous messages andinhibit translation. Since ribozymes unlike antisense molecules, arecatalytic, a lower intracellular concentration is required forefficiency.

[0409] Antagonist/agonist compounds may be employed to inhibit the cellgrowth and proliferation effects of the polypeptides of the presentinvention on neoplastic cells and tissues, i.e. stimulation ofangiogenesis of tumors, and, therefore, retard or prevent abnormalcellular growth and proliferation, for example, in tumor formation orgrowth.

[0410] The antagonist/agonist may also be employed to preventhyper-vascular diseases, and prevent the proliferation of epitheliallens cells after extracapsular cataract surgery. Prevention of themitogenic activity of the polypeptides of the present invention may alsobe desirous in cases such as restenosis after balloon angioplasty.

[0411] The antagonist/agonist may also be employed to prevent the growthof scar tissue during wound healing.

[0412] The antagonist/agonist may also be employed to treat, prevent,and/or diagnose the diseases described herein.

[0413] Thus, the invention provides a method of treating or preventingdiseases, disorders, and/or conditions, including but not limited to thediseases, disorders, and/or conditions listed throughout thisapplication, associated with overexpression of a polynucleotide of thepresent invention by administering to a patient (a) an antisensemolecule directed to the polynucleotide of the present invention, and/or(b) a ribozyme directed to the polynucleotide of the present invention.

[0414] Chromosome

[0415] Assays

[0416] Chromosome mapping studies have shown that the BAIT gene maps inthe human genome to the location 4q31.2-31.3. Thus, the nucleic acidmolecules of the present invention are also valuable for chromosomeidentification. The sequence is specifically targeted to and canhybridize with the above particular location on an individual humanchromosome. Moreover, there is a current need for identifying particularsites on the chromosome. Few chromosome marking reagents based on actualsequence data (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

[0417] In certain preferred embodiments in this regard, the cDNA hereindisclosed is used to clone genomic DNA of a BAIT protein gene. This canbe accomplished using a variety of well known techniques and libraries,which generally are available commercially. The genomic DNA then is usedfor in situ chromosome mapping using well known techniques for thispurpose. Typically, in accordance with routine procedures for chromosomemapping, some trial and error may be necessary to identify a genomicprobe that gives a good in situ hybridization signal.

[0418] In addition, in some cases, sequences can be mapped tochromosomes by preparing PCR primers (preferably 15-25 bp) from thecDNA. Computer analysis of the 3 untranslated region of the gene is usedto rapidly select primers that do not span more than one exon in thegenomic DNA, thus complicating the amplification process. These primersare then used for PCR screening of somatic cell hybrids containingindividual human chromosomes. Only those hybrids containing the humangene corresponding to the primer will yield an amplified portion.

[0419] PCR mapping of somatic cell hybrids is a rapid procedure forassigning a particular DNA to a particular chromosome. Using the presentinvention with the same oligonucleotide primers, sublocalization can beachieved with panels of portions from specific chromosomes or pools oflarge genomic clones in an analogous manner. Other mapping strategiesthat can similarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

[0420] Fluorescence in situ hybridization (“FISH”) of a cDNA clone to ametaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with probesfrom the cDNA as short as 50 or 60 bp. For a review of this technique,see Verma et al., Human Chromosomes: A Manual Of Basic Techniques,Pergamon Press, New York (1988).

[0421] Once a sequence has been mapped to a precise chromosomallocation, the physical position of the sequence on the chromosome can becorrelated with genetic map data. Such data are found, for example, inV. McKusick, Mendelian Inheritance In Man, available on-line throughJohns Hopkins University, Welch Medical Library. The relationshipbetween genes and diseases that have been mapped to the same chromosomalregion are then identified through linkage analysis (coinheritance ofphysically adjacent genes).

[0422] Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

[0423] With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. This assumes I megabase mapping resolution and one geneper 20 kb.

EXAMPLES Example 1 Expression and Purification of BAIT in E. coli

[0424] Having generally described the invention, the same will be morereadily understood by reference to the following examples, which areprovided by way of illustration and are not intended as limiting.

[0425] The bacterial expression vector pQE9 (pD 10) was used forbacterial expression in this example. (QIAGEN, Inc., 9259 Eton Avenue,Chatsworth, Calif., 91311). pQE9 encodes ampicillin antibioticresistance (“Ampr”) and contains a bacterial origin of replication(“ori”), an IPTG inducible promoter, a ribosome binding site (“RBS”),six codons encoding histidine residues that allow affinity purificationusing nickel-nitrilo-triacetic acid (“Ni-NTA”) affinity resin sold byQIAGEN, Inc., supra, and suitable single restriction enzyme cleavagesites. These elements are arranged such that an inserted DNA fragmentencoding a polypeptide expresses that polypeptide with the six Hisresidues (i.e., a “6× His tag”) covalently linked to the amino terminusof that polypeptide.

[0426] The DNA sequence encoding the desired portion BAIT proteinlacking the hydrophobic leader sequence was amplified from the depositedcDNA clone using PCR oligonucleotide primers which anneal to the aminoterminal sequences of the desired portion of the BAIT protein and tosequences in the deposited construct 3′ to the cDNA coding sequence.Additional nucleotides containing restriction sites to facilitatecloning in the pQE9 vector are added to the 5′ and 3′ primer sequences,respectively.

[0427] For cloning the mature protein, the 5′ primer has the sequence 5′GAGCATGGATCCGCCACTTTCCCTGAGGAA 3′ (SEQ ID NO:10) containing theunderlined Bam-HI restriction site followed by 18 nucleotides of theamino terminal coding sequence of the mature BAIT sequence in FIG. 1.One of ordinary skill in the art would appreciate, of course, that thepoint in the protein coding sequence where the 5′ primer begins may bevaried to amplify a DNA segment encoding any desired portion of thecomplete BAIT protein shorter or longer than the mature form. The 3′primer has the sequence 5′ GCACATGGATCCTTAAAGTTCTTCGAAATCATG 3′ (SEQ IDNO:11) containing the underlined BamHI restriction site followed by 21nucleotides complementary to the 3′ end of the coding sequence of theBAIT DNA sequence in FIG. 1.

[0428] The amplified BAIT DNA fragment and the vector pQE9 were digestedwith BamHl and the digested DNAs are then ligated together. Insertion ofthe BAIT DNA into the restricted pQE9 vector places the BAIT proteincoding region downstream from the IPTG-inducible promoter and in-framewith an initiating AUG and the six histidine codons.

[0429] The ligation mixture was transformed into competent E. coli cellsusing standard procedures such as those described in Sambrook et al.,Molecular Cloning: a Laboratory Manual, 2nd Ed.; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989). E. coli strainM15/rep4, containing multiple copies of the plasmid pREP4, whichexpresses the lac repressor and confers kanamycin resistance (“Kanr”),is used in carrying out the illustrative example described herein. Thisstrain, which is only one of many that are suitable for expressing BAITprotein, is available commercially from QIAGEN, Inc., supra.Transformants were identified by their ability to grow on LB plates inthe presence of ampicillin and kanamycin. Plasmid DNA was isolated fromresistant colonies and the identity of the cloned DNA confirmed byrestriction analysis, PCR and DNA sequencing.

[0430] Clones containing the desired constructs were grown overnight(“O/N”) in liquid culture in LB media supplemented with both ampicillin(100 μg/ml) and kanamycin (25 μg/ml). The O/N culture is used toinoculate a large culture, at a dilution of approximately 1:25 to 1:250.The cells were grown to an optical density at 600 nm (“OD600”) ofbetween 0.4 and 0.6 isopropyl-b-D-thiogalactopyranoside (“IPTG”) wasthen added to a final concentration of 1 mM to induce transcription fromthe lac repressor sensitive promoter, by inactivating the lacdrepressor. Cells subsequently were incubated further for 3 to 4 hours.Cells then were harvested by centrifugation.

[0431] The cells were then stirred for 3-4 hours at 4° C. in 6Mguanidine-HCl, pH 8. The cell debris was removed by centrifugation, andthe supernatant containing the BAIT was loaded onto anickel-nitrilo-tri-acetic acid (“Ni-NTA”) affinity resin column(available from QIAGEN, Inc., supra). Proteins with a 6× His tag bind tothe NiNTA resin with high affinity and can be purified in a simpleone-step procedure (for details see: The QlAexpressionist, 1995, QIAGEN,Inc., supra). Briefly the supenatant is loaded onto the column in 6 Mguanidine-HCl, pH 8, the column is first washed with 10 volumes of 6 Mguanidine-HCl, pH 8, then washed with 10 volumes of 6 M guanidine-HCl pH6, and finally the BAIT is eluted with 6 M guanidine-HCl, pH 5.

[0432] The purified protein was then renatured by dialyzing it againstphosphate buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus200 mM NaCl. Alternatively, the protein can be successfully refoldedwhile immobilized on the Ni-NTA column. The recommended conditions areas follows: renature using a linear 6M-1 M urea gradient in 500 mM NaCl,20% glycerol, 20 mM Tris/HCl pH 7.4, containing protease inhibitors. Therenaturation should be performed over a period of 1.5 hours or more.After renaturation the proteins can be eluted by the addition of 250 mMimidazole. Immidazole is removed by a final dialyzing step against PBSor 50 mM sodium acetate pH 6 buffer plus 200 miM NaCl. The purifiedprotein is stored at 4° C. or frozen at −80° C.

Example 2 Cloning, Expression and Purification of BAIT Protein in aBaculovirus Expression System

[0433] In this illustrative example, the plasmid shuttle vector pA2 isused to insert the cloned DNA encoding the complete protein, includingits naturally associated secretory signal (leader) sequence, into abaculovirus to express the mature BAIT protein, using standard methodsas described in Summers et al., A Manual of Methods for BaculovirusVectors and Insect Cell Culture Procedures, Texas AgriculturalExperimental Station Bulletin No. 1555 (1987). This expression vectorcontains the strong polyhedrin promoter of the Autographa californicanuclear polyhedrosis virus (AcMINPV) followed by convenient restrictionsites such as BamHl, Xba I and Asp718. The polyadenylation site of thesimian virus 40 (“SV40”) is used for efficient polyadenylation. For easyselection of recombinant virus, the plasmid contains thebeta-galactosidase gene from E. coli under control of a weak Drosophilapromoter in the same orientation, followed by the polyadenylation signalof the polyhedrin gene. The inserted genes are flanked on both sides byviral sequences for cell-mediated homologous recombination withwild-type viral DNA to generate viable virus that express the clonedpolynucleotide.

[0434] Many other baculovirus vectors could be used in place of thevector above, such as pAc373, pVL941 and pAcIM1, as one skilled in theart would readily appreciate, as long as the construct providesappropriately located signals for transcription, translation, secretionand the like, including a signal peptide and an in-frame AUG asrequired. Such vectors are described, for instance, in Luckow et al.,Virology 170:31-39 (1989).

[0435] The cDNA sequence encoding the full length BAIT protein in thedeposited clone, including the AUG initiation codon and the naturallyassociated leader sequence shown in FIG. 1 (SEQ ID NO:2), is amplifiedusing PCR oligonucleotide primers corresponding to the 5′ and 3′sequences of the gene. The 5′ primer has the sequence 5′GAGCATGGATCCGCCATCATGGCTTTCCTTGGACTC 3′ (SEQ ID NO:12) containing theunderlined BamHI restriction enzyme site, an efficient signal forinitiation of translation in eukaryotic cells, as described by Kozak,M., J. Mol. Biol. 196:947-950 (1987), followed by 18 nucleotides of thesequence of the complete BAIT protein shown in FIG. 1, beginning withthe AUG initiation codon. The 3′ primer has the sequence5′-GAGCATTCTAGAGTTGCAAACATAATGTGC-3′ (SEQ ID NO:13) containing theunderlined XbaI restriction site followed by 18 nucleotidescomplementary to the 3′ noncoding sequence in FIG. 1.

[0436] The amplified fragment was isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment then is digested with BamHI and XbaI and again waspurified on a 1% agarose gel. This fragment is designated herein F1.

[0437] The plasmid was digested with the restriction enzymes BamHI andXbaI using routine procedures known in the art. The DNA was thenisolated from a 1% agarose gel using a commercially available kit(“Geneclean” BIO 101 Inc., La Jolla, Calif.). This vector DNA isdesignated herein “V1”.

[0438] Fragment F1 and the plasmid VI were ligated together with T4 DNAligase. Competent E. coli cells were transformed with the ligationmixture and spread on culture plates. Bacteria were identified thatcontain the plasmid with the human BAIT gene by digesting DNA fromindividual colonies using BamHI and XbaI and then analyzing thedigestion product by gel electrophoresis. The sequence of the clonedfragment was confirmed by DNA sequencing. This plasmid is designatedherein pA2BAIT.

[0439] Five μg of the plasmid pA2BAIT was co-transfected with 1.0 μg ofa commercially available linearized baculovirus DNA (“BaculoGold™baculovirus DNA”, Pharmingen, San Diego, Calif.), using the lipofectionmethod described by Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987). One μg of BaculoGold™ virus DNA and 5 μg of theplasmid pA2BAIT were mixed in a sterile well of a microtiter platecontaining 50 μl of serum-free Grace's medium (Life Technologies Inc.,Gaithersburg, Md.). Afterwards, 10 μl Lipofectin plus 90 μl Grace'smedium were added, mixed and incubated for 15 minutes at roomtemperature. Then the transfection mixture was added drop-wise to Sf9insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with1 ml Grace's medium without serum. The plate was then incubated for 5hours at 27° C. After 5 hours the transfection solution was removed fromthe plate and 1 ml of Grace's insect medium supplemented with 10% fetalcalf serum was added. The plate was put back into an incubator andcultivation was continued at 27° C. for four days.

[0440] After four days the supenatant was collected and a plaque assaywas performed, as described by Summers and Smith, supra. An agarose gelwith “Blue Gal” (Life Technologies Inc., Gaithersburg) was used to alloweasy identification and isolation of gal-expressing clones, whichproduce blue-stained plaques. (A detailed description of a “plaqueassay” of this type can also be found in the user's guide for insectcell culture and baculovirology distributed by Life Technologies Inc.,Gaithersburg, page 9-10). After appropriate incubation, blue stainedplaques were picked with the tip of a micropipettor (e.g., Eppendorf).The agar containing the recombinant viruses were then resuspended in amicrocentrifuge tube containing 200 μl of Grace's medium and thesuspension containing the recombinant baculovirus was used to infect Sf9cells seeded in 35 mm dishes. Four days later the supernatants of theseculture dishes were harvested and then they were stored at 4° C. Therecombinant virus is called V-BAIT.

[0441] To verify the expression of the BAIT gene Sf9 cells were grown inGrace's medium supplemented with 10% heat-inactivated FBS. The cellswere infected with the recombinant baculovirus V-BAIT at a multiplicityof infection (“MOI”) of about 2. Six hours later the medium was removedand replaced with SF900 II medium minus methionine and cysteine(available from Life Technologies Inc., Rockville, Md.). 42 hours later,5 μCi of ³⁵S-methionine and 5 μCi ³⁵S-cysteine (available from Amersham)were added. The cells were further incubated for 16 hours and thenharvested by centrifugation. The proteins in the supernatant as well asthe intracellular proteins were analyzed by SDS-PAGE followed byautoradiography (if radiolabeled).

[0442] For production of unlabeled BAIT polypeptide, Sf9 cells wereseeded in serum-free media at a density of 1.5×10⁶ cells/ml in 200 mlspinner flasks. They were infected at an multiplicity of infection (moi)of 1 with the recombinant baculovirus encoding BAIT. At 96 hrspost-infection (pi), the cells were removed by centrifugation, and theconditioned media used as starting material.

[0443] Medium was diluted 1:1 (vol:vol) with 50 mM Na-Acetate PH 6.0(Buffer A). The sample was applied to an HQ-50 column (Poros Resins,Perseptive Biosystems) at a flow rate of 30 mls/min. Bound protein wasstep-eluted with Buffer A containing 0.15, 0.35, 0.6 and 1.0 M NaCl andthe fractions analyzed by SDS-PAGE. BAIT-containing fraction (350 mMstep) were pooled, and diluted with Buffer A to a final NaClconcentration of 50 mM. This sample was applied to an HS-50 column(Poros Resins, Perseptive Biosystems) previously equilibrated withBuffer A plus 50 mM NaCl at a flow rate of 10 mls/min. Bound proteinswere step eluted with Buffer A containing 1.0 M NaCl and fractionsanalyzed by SDS-PAGE. Finally, the pooled fractions were applied to anS-200 (Pharmacia) gel filtration column previously equilibrated with 50mM Na-Acetate pH 6.5; 250 mM NaCl. BAIT-containing fractions eluted as asingle peak which were pooled.

[0444] Protein concentration was determined using the Bio-Rad ProteinAssay with BSA as a standard. Alternatively, the BCA Assay (Pierce) wasused. The protein was 90% pure as judged by SDS-PACE. The baculovirusproduced protein was shown to be glycosylated and the isolectric point(pI) of the protein was determined to be 5.0. This protein was used forin vitro activity assays described hereinabove. Microsequencing of theamino acid sequence of the amino terminus of the purified proteinimmediately after purification was used to determine the amino terminalsequence of the mature protein and thus the cleavage point and length(18 amino acids) of the secretory signal peptide, as shown in FIG. 1(SEQ ID NO:2). However, subsequent sequencing of the same preparation inanother laboratory following storage at −80° C. for several weeksrevealed an approximately equal molar mixture of the original maturespecies and a second species lacking one additional residue, i.e., withthe N terminus ending with Thr at position 19 (and thus comprising aminoacids 19-410 of SEQ ID NO:2). Both species appeared to be efficientlycleaved upon interaction with tPA.

Example 3 Cloning and Expression in Mammalian Cells

[0445] A typical mammalian expression vector contains the promoterelement, which mediates the initiation of transcription of mRNA, theprotein coding sequence, and signals required for the termination oftranscription and polyadenylation of the transcript. Additional elementsinclude enhancers, Kozak sequences and intervening sequences flanked bydonor and acceptor sites for RNA splicing. Highly efficienttranscription can be achieved with the early and late promoters fromSV40, the long terminal repeats (LTRs) from Retroviruses, e.g., RSV,HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV).However, cellular elements can also be used (e.g., the human actinpromoter). Suitable expression vectors for use in practicing the presentinvention include, for example, vectors such as pSVL and pMSG(Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC37146) and pBC12MI (ATCC 67109). Mammalian host cells that could be usedinclude, human Hela, 293, H9 and Jurkat cells, mouse NIH3T3 and C 127cells, Cos 1, Cos 7 and CV1, quail QC1-3 cells, mouse L cells andChinese hamster ovary (CHO) cells.

[0446] Alternatively, the gene can be expressed in stable cell linesthat contain the gene integrated into a chromosome. The co-transfectionwith a selectable marker such as dhfr, gpt, neomycin, hygromycin allowsthe identification and isolation of the transfected cells.

[0447] The transfected gene can also be amplified to express largeamounts of the encoded protein. The DHFR (dihydrofolate reductase)marker is useful to develop cell lines that carry several hundred oreven several thousand copies of the gene of interest. Another usefulselection marker is the enzyme glutamine synthase (GS) (Murphy et al.,Biochem J. 22 7:277-279 (1991); Bebbington et al., BiolTechnology 10:169-175 (1992)). Using these markers, the mammalian cells are grown inselective medium and the cells with the highest resistance are selected.These cell lines contain the amplified gene(s) integrated into achromosome. Chinese hamster ovary (CHO) and NSO cells are often used forthe production of proteins.

[0448] The expression vectors pC1 and pC4 contain the strong promoter(LTR) of the Rous Sarcoma Virus (Cullen et al., Molecular and CellularBiology, 438-447 (March, 1985)) plus a fragment of the CMV-enhancer(Boshart et al., Cell 41:521-530 (1985)). Multiple cloning sites, e.g.,with the restriction enzyme cleavage sites BanHI, XbaI and Asp718,facilitate the cloning of the gene of interest. The vectors contain inaddition the 3′ intron, the polyadenylation and termination signal ofthe rat preproinsulin gene.

Example 3(a) Cloning and Expression in COS Cells

[0449] The expression plasmid, pBAIT HA, is made by cloning a cDNAencoding BAIT into the expression vector pcDNAI/Amp or pcDNAIII (whichcan be obtained from Invitrogen, Inc.).

[0450] The expression vector pcDNAI/amp contains: (1) an E. coli originof replication effective for propagation in E. coli and otherprokaryotic cells; (2) an ampicillin resistance gene for selection ofplasmid-containing prokaryotic cells; (3) an SV40 origin of replicationfor propagation in eukaryotic cells; (4) a CMV promoter, a polylinker,an SV40 intron; (5) several codons encoding a hemagglutinin fragment(i.e., an “HA” tag to facilitate purification) followed by a terminationcodon and polyadenylation signal arranged so that a cDNA can beconveniently placed under expression control of the CMV promoter andoperably linked to the SV40 intron and the polyadenylation signal bymeans of restriction sites in the polylinker. The HA tag corresponds toan epitope derived from the influenza hemagglutinin protein described byWilson et al., Cell 3-7:767 (1984). The fusion of the HA tag to thetarget protein allows easy detection and recovery of the recombinantprotein with an antibody that recognizes the HA epitope. pcDNAIRcontains, in addition, the selectable neomycin marker.

[0451] A DNA fragment encoding the BAIT is cloned into the polylinkerregion of the vector so that recombinant protein expression is directedby the CMV promoter. The plasmid construction strategy is as follows.The BAIT cDNA of the deposited clone is amplified using primers thatcontain convenient restriction sites, much as described above forconstruction of vectors for expression of BAIT in E. coli. Suitableprimers include the following, which are used in this example. The 5′primer, containing the underlined BamHI site, a Kozak sequence. an AUGstart codon and 18 nucleotides of the 5′ coding region of the completeBAIT has the following sequence: 5′ GAGCATGGATCCGCCATCATGGCTTTCCTTGGACTC3′ (SEQ ID NO:14). The 3′ primer, containing the underlined BamHI siteand 15 nucleotides complementary to the 3′ coding sequence, has thefollowing sequence: 5′ GCACATGGATCCAAGTTCTTCGAAATCATG 3′ (SEQ ID NO:15).

[0452] The PCR amplified DNA fragment and the vector, pcDNAI/Amp, aredigested with BamHI, the vector is dephosphorylated and then the vectorand amplified DNA are ligated. The ligation mixture is transformed intoE. coli strain SURE (available from Stratagene Cloning Systems, 11099North Torrey Pines Road, La Jolla, Calif. 92037), and the transformedculture is plated on ampicillin media plates which then are incubated toallow growth of ampicillin resistant colonies. Plasmid DNA is isolatedfrom resistant colonies and examined by restriction analysis or othermeans for the presence of the BAIT-encoding fragment.

[0453] For expression of recombinant BAIT, COS cells are transfectedwith an expression vector, as described above, using DEAE-DEXTRAN, asdescribed, for instance, in Sambrook et al., Molecular Cloning: aLaboratory Manual, Cold Spring Laboratory Press, Cold Spring Harbor,N.Y. (1989). Cells are incubated under conditions for expression of BAITby the vector.

[0454] Expression of the BAIT-HA fusion protein is detected byradiolabeling and immunoprecipitation, using methods described in, forexample Harlow et al., Antibodies. A Laboratory Manual, 2nd Ed.; ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988). To thisend, two days after transfection, the cells are labeled by incubation inmedia containing ³⁵-cysteine for 8 hours. The cells and the media arecollected, and the cells are washed and the lysed with detergent-containing RIPA buffer: 150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5%DOC, 50 mM TRIS, pH 7.5, as described by Wilson et al. cited above.Proteins are precipitated from the cell lysate and from the culturemedia using an HA-specific monoclonal antibody. The precipitatedproteins then are analyzed by SDS-PAGE and autoradiography. Anexpression product of the expected size is seen in the cell lysate,which is not seen in negative controls.

Example 3(b) Cloning and Expression in CHO Cells

[0455] The vector pC4 is used for the expression of BAIT protein.Plasmid pC4 is a derivative of the plasmid pSV2-dhfr (ATCC Accession No.37146) The plasmid contains the mouse DHFR gene under control of theSV40 early promoter. Chinese hamster ovary- or other cells lackingdihydrofolate activity that are transfected with these plasmids can beselected by growing the cells in a selective medium (alpha minus MEM,Life Technologies) supplemented with the chemotherapeutic agentmethotrexate. The amplification of the DHFR genes in cells resistant tomethotrexate (MTX) has been well documented (see, e.g., Alt, F. W.,Kellems, R. M., Bertino, J. R., and Schimke, R. T., 1978, J. Biol. Chem.253:1357-1370, Hamlin, J. L. and Ma, C. 1990, Biochem. et Biophys. Acta,1097:107-143, Page, M. J. and Sydenham, M. A. 1991, Biotechnology9:64-68). Cells grown in increasing concentrations of MTX developresistance to the drug by overproducing the target enzyme, DHFR, as aresult of amplification of the DHFR gene. If a second gene is linked tothe DHFR gene, it is usually co-amplified and over-expressed. It isknown in the art that this approach may be used to develop cell linescarrying more than 1,000 copies of the amplified gene(s). Subsequently,when the methotrexate is withdrawn, cell lines are obtained whichcontain the amplified gene integrated into one or more chromosome(s) ofthe host cell.

[0456] Plasmid pC4 contains for expressing the gene of interest thestrong promoter of the long terminal repeat (LTR) of the Rouse SarcomaVirus (Cullen, et al., Molecular and Cellular Biology, March1985:438-447) plus a fragment isolated from the enhancer of theimmediate early gene of human cytomegalovirus (CMV) (Boshart et al.,Cell 41:521-530 (1985)). Downstream of the promoter are the followingsingle restriction enzyme cleavage sites that allow the integration ofthe genes: BamHI, XbaI, and Asp718. Behind these cloning sites theplasmid contains the 3′ intron and polyadenylation site of the ratpreproinsulin gene. Other high efficiency promoters can also be used forthe expression, e.g., the human β-actin promoter, the SV40 early or latepromoters or the long terminal repeats from other retroviruses, e.g.,HIV and HTLVI. Clontech's Tet-Off and Tet-On gene expression systems andsimilar systems can be used to express the BAIT in a regulated way inmammalian cells (Gossen, M., & Bujard, H. 1992, Proc. Natl. Acad. Sci.USA 89: 5547-5551). For the polyadenylation of the MRNA other signals,e.g., from the human growth hormone or globin genes can be used as well.Stable cell lines carrying a gene of interest integrated into thechromosomes can also be selected upon co-transfection with a selectablemarker such as gpt, G418 or hygromycin. It is advantageous to use morethan one selectable marker in the beginning, e.g., G418 plusmethotrexate.

[0457] The plasmid pC4 is digested with the restriction enzymes BamHIand XbaI and then dephosphorylated using calf intestinal phosphates byprocedures known in the art. The vector is then isolated from a 1%agarose gel. J. Mol. Biol. 196:947-950 (1987), and 24 bases of thecoding sequence of BAIT shown in FIG. 1 (SEQ ID NO:1). The 3′ primer hasthe sequence 5′ GAGCATTCTAGAGTTGCAAACATAATGTGC 3′ (SEQ ID NO:17)containing the underlined XbaI restriction site followed by 18nucleotides complementary to the non-translated region of the BAIT geneshown in FIG. 1 (SEQ ID NO:1).

[0458] The amplified fragment is digested with the endonucleases BamHIand XbaI and then purified again on a 1% agarose gel. The isolatedfragment and the dephosphorylated vector are then ligated with T4 DNAligase. E. coli HB101 or XL-1 Blue cells are then transformed andbacteria are identified that contain the fragment inserted into plasmidpC4 using, for instance, restriction enzyme analysis.

[0459] Chinese hamster ovary cells lacking an active DHFR gene are usedfor transfection. Five μg of the expression plasmid pC4 is cotransfectedwith 0.5 μg of the plasmid pSVneo using lipofectin (Felgner et al.,supra). The plasmid pSV2-neo contains a dominant selectable marker, theneo gene from Tn5 encoding an enzyme that confers resistance to a groupof antibiotics including G418. The cells are seeded in alpha minus MEMsupplemented with 1 mg/ml G418. After 2 days, the cells are trypsinizedand seeded in hybridoma cloning plates (Greiner, Germany) in alpha minusMEM supplemented with 10, 25, or 50 ng/ml of metothrexate plus 1 mg/mlG418. After about 10-14 days single clones are trypsinized and thenseeded in 6-well petri dishes or 10 ml flasks using differentconcentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM).Clones growing at the highest concentrations of methotrexate are thentransferred to new 6-well plates containing even higher concentrationsof methotrexate (1 μM, 2 μM, 5 uM, 10 mM, 20 mM). The same procedure isrepeated until clones are obtained which grow at a concentration of100-200 μM. Expression of the desired gene product is analyzed, forinstance, by SDS- PAGE and Western blot or by reversed phase HPLCanalysis.

Example 4 Tissue Distribution of BAIT Protein Expression

[0460] Northern blot analysis is carried out to examine BAIT geneexpression in human tissues, using methods described by, among others,Sambrook et al., cited above. A cDNA probe containing the entirenucleotide sequence of the BAIT protein (SEQ ID NO:1) is labeled with³²P using the rediprime ™DNA labeling system (Amersham Life Science),according to manufacturer's instructions. After labeling, the probe ispurified using a CHROMA SPIN-100™ column (Clontech Laboratories, Inc.),according to manufacturer's protocol number PT1200-1. The purifiedlabeled probe is then used to examine various human tissues for BAITmRNA.

[0461] Multiple Tissue Northern (MTN) blots containing various humantissues (H) or human immune system tissues (IM) are obtained fromClontech and are examined with the labeled probe using ExpressHyb™hybridization solution (Clontech) according to manufacturer's protocolnumber PT1190-1. Following hybridization and washing, the blots aremounted and exposed to film at −70 C overnight, and films developedaccording to standard procedures.

Example 5 Immunohistochemical Analysis of BAIT

[0462] To more precisely examine the expression of BAIT protein,immunohistochemical staining of adult mouse tissue sections wasperformed. Consistent with the mRNA distribution only brain and spinalcord tissues demonstrated significant reactivity. BAIT is widelydistributed throughout the brain, but is primarily localized to neurons.The major exceptions to this pattern are expression in the ependymalcells of the choroid plexus, and the brush border of the cells liningthe ventricles. These cells are thought to be of Microglial origin areimportant for maintaining the cerebrospinal and ventricular fluid. Otherregions of the brain with high BAIT immunoreactivity are the Purkinjicells of the cerebellum which show strongly positive staining of thecell body as well as the axons. Most neurons of the spinal cord are alsostrongly positive, as are the axons in and the myelinated tracts of thecommissura. Another region of strong staining is the hypothalamus wheremost of the neurons appear to show significant amounts of BAITimmunoreactivity within the cell body. Finally, BAIT was present in thelarge motor neurons of the medulla oblongata and in scattered neuronsthroughout the cortex.

Example 6 Comparison of BAIT Inhibitory Activity

[0463] A comparison of BAIT inhibitory activity and expression with thatof PAI-I and PN-1, suggests that BAIT has a biological role distinctfrom these other serpins. While BAIT reacts about 30-fold slower withtPA than does PAI-1, its rate of 6.2×10⁻⁵ M⁻¹s⁻¹ it is about 20-foldfaster than that of PN-1. In addition, BAIT's primary target enzymeappears to be tPA, since its rate of inhibition of tPA is approximately25-fold faster than is its rate of inhibition of uPA (Table 1). Incontrast, PAI-I inhibits uPA and tPA with essentially the same ratewhile PN-1 reacts with uPA about 5-fold faster than it does with tPA.Finally, unlike PAI-1 and PN-1, BAIT's inhibition of thrombin is notstimulated by heparin. Table I describes the kinetic constants for theinteraction of BAIT with various proteinases. TABLE I Enzyme k (s⁻¹)K(μM) ki(M⁻¹s⁻¹) tctPA 0.078 0.12 6.2 × 10⁵ sctPA 0.17 2.1 8.0 × 10⁴Trypsin 0.0096 0.16 5.9 × 10⁴ uPA-H 0.0050 0.20 2.5 × 10⁴ uPA-L 0.0131.4 9.2 × 10³ NGF-_(γ) 0.0086 1.3 6.5 × 10³ PlasmiN 0.000052 0.15 3.6 ×10² Thrombin 0.000131 0.64 2.1 × 10²

[0464] The abbreviations are tctPA, human two-chain tPA: sctPA, humansingle-chain tPA, uPA-H, human high molecular weight uPA; uPA-L, humanlow molecular weight uPA; trypsin, bovine beta trypsin; and NGF-gamma,rat nerve growth factor gamma.

Example 7 BAIT Activity in Stoke Models

[0465] The present study demonstrates that BAIT is expressed in the areaof ischemic penumbra in an animal model of focal cerebralischemia/reperfusion. Moreover, intracerebral administration of BAITafter stroke decreases stroke volume, reduces basement membraneproteolysis, and diminishes the number of cells with apoptotic featuresin the area of ischemic penumbra. Thus, the data presented suggest thatBAIT, a selective, naturally occurring inhibitor of tPA, may play animportant role in neuronal survival after stroke.

[0466] Animal Preparation and Surgery:

[0467] Adult male Sprague-Dawley rats weighing 350-400 g were used.Anesthesia was induced with 4% halothane, 70% nitrous oxide and abalance of oxygen, and was maintained with 2% halothane and 70% nitrousoxide during the surgical procedure. Rats were intubated endotracheallyand mechanically ventilated. Arterial blood pressure and blood gaseswere monitored. Body temperature was maintained at 37.5±0.5° C. with awarming blanket (Animal Blanket Control Unit, Harvard Apparatus) andcontrolled with a rectal thermistor and a probe inserted into themasseter muscle. Middle cerebral artery (MCA) was exposed and cauterizedwith a microbipolar coagulator (Non-Stick Bipolar Coagulation Forceps,Kirwan Surgical Products, Marshfield, Mass.) above its crossing pointwith the inferior cerebral vein as described elsewhere. Tamura A, et.al. J Cereb.Blood Flow Metab. 1981;1:53-60. Animals were then placed ona stereotactic frame and 20 μl of either 30 μM active BAIT in PBS, 30 μMinactive elastase-cleaved BAIT in PBS or 20 μl of PBS only was injectedintracortically with a Hamilton Syringe through the burr hole.Comparison of untreated animals (no injection) to PBS-treated ratsindicated that there was no significant difference in stroke volume,indicating that the injection itself did not contribute to the infarctsize (data not shown). Following the intracortical injections, the leftcommon carotid artery was exposed through a midline cervical incisionand temporarily occluded for one hour with a microaneurysm clip (8 mm,100 g pressure; Roboz Surgical Instruments Co., Rockville, MD). Brint S,et. al. J Cereb.Blood Flow Metab. 1988;8:474-485. Animals where thenallowed to recover under the heating lamp, returned to their cages andgiven free access to water.

[0468] Infarct Volume:

[0469] Rats were anesthetized with pentobarbital i.p. 72 hours afterinfarction and brains were removed after transcardiac perfusion with PBSand parafomaldehyde 4% (Fisher Scientific, HC-200). The entire brain wasembedded in paraffin and coronal sections 20 μm thick, were cut throughthe rostrocaudal extent of the brain (FIG. 6). The sections were stainedwith hematoxilin-eosin and using the NIH Image Analyzer System, thetotal volume of each infarction was determined by the integration of theareas of eight chosen sections and the distances between them. Therostral and caudal limits for the integration were set at the frontaland occipital poles of the cortex. Osborne K A, et. al., JNeurol.Neurosurg.Psychiatry 1987;50:402-410. Statistical significancebetween groups of animals was identified by a Student's t-test.

[0470] TUNEL Staining:

[0471] Five μm paraffin-embedded sections from BAIT- and control-treatedanimals sacrificed at 6, 24, 48, and 72 hours after reperfusion wereexamined for TUNEL reactivity using the Apoptag Kit (Oncor, Gaithesburg,MD). Paraffin sections were dewaxed, rehydrated and treated withproteinase K (20 μg/ml), and blocked for endogenous peroxidase activitywith 3% H₂O₂. Subsequent end-labeling was done with TdT enzyme at 37° C.for 1 hour. Anti-digoxigenin peroxidase conjugate was applied to thetissue for 30 minutes at room temperature. The slides were developedwith peroxidase substrate DAB for 5 minutes (Sigma, St. Louis, MO),washed in dH₂O for 5 minutes and counter-stained with 0.5% methyl greenfor 10 minutes. To quantitate the presence of cells with apoptoticbodies, an area surrounding the ischemic core extending from thecerebral cortex to the most anterior (septal) part of the hippocampuswas imaged in BAIT- and control-treated animals. Histologic featuresused by light microscopy to identify apoptosis depended upon recognitionof dark-brown rounded or oval apoptotic bodies. MacManus J P, et. al.,Neurosci.Lett. 1993;164:89-92; Li Y, et. al., Am.J Pathol.1995;146:1045-1051. Statistical significance between groups of animalswas identified by a Student's t-test.

[0472] Zymography:

[0473] For SDS-PAGE zymography the region containing the stroke inbrains from BAIT- and PBS-treated animals sacrificed at 6 and 72 hoursafter reperfusion were dissected and slices of approximately 600 mg werefrozen in dry ice and stored at −70° C. A similar portion of brain wasdissected from the same area in the contralateral hemisphere in bothBAIT-treated and control animals. Protein extracts were prepared in 1.2ml of extraction buffer as described. Hastings G A, et. al., JBiol.Chem. 1997;272:33062-33067. The protein concentration was thendetermined, and 30 μg of extract (approximately 1 μl) was mixed withnon-reducing sample buffer and subjected to SDS-PAGE on a 10% gel(Novex, San Diego, Calif.). Human tPA 0.3 ng (Genentech, San Francisco,Calif.) and a rat kidney extract containing uPA prepared in the same wayas the brain extracts were included as positive controls and theidentity of each PA was determined by including either anti-tPA oranti-uPA in the indicator film (data not shown). Followingelectrophoresis, the gel was soaked in 2.5% Triton X-100 for 2×45 min toremove the SDS. An indicator gel was prepared by mixing 1.25 ml of an 8%solution of boiled and centrifuged milk in PBS, 5 ml PBS and 3.75 ml ofa 2.5% agar solution prewarmed at 50° C. Plasminogen (MolecularInnovations, Royal Oak, Mich.), was added to a final concentration of 30μg/ml and the solution mixed and poured onto a pre-warmed glass plate.The Triton X-100 soaked gel was applied to the surface of theplasminogen-milk indicator gel and incubated in a humid chamber at 37°C. Milk indicator gel without plasminogen was also included as acontrol. The relative increase of tPA and uPA ipsilateral to the strokeat 6 hours after reperfusion was quantified by scanning a photograph ofthe SDS-PAGE zymography gel taken at an early time of development,before full lysis had occurred, and using the NIH Image Analyzer System.Normal baseline PA activities were calculated from the average of theactivity present in 6 independent contralateral samples for which thecoefficient of variation was <0.2%. Control analysis of purified tPA bythis method demonstrated that lysis was linear over at least an 8-foldrange with a Correlation Coefficient (r) of 0.994. Statisticalsignificance between groups was identified by a Student's t-test.

[0474] For the in situ proteinase activity assay, brains from BAIT- andcontrol-treated animals sacrificed at 6 and 72 hours after reperfusion(n=3 for each condition at each time point) were frozen in OCT andstored at −70° C. Eight μm cryostat sections were examined forplasminogen activator activity in overlays prepared as described.Sappino A P, et al., J.Clin.Invest. 1993;92:679-685. One hundred fiftyμl of the overlay mixture was applied to pre-warmed tissue sections andspread under glass cover slips. Slides were incubated in a humid chamberat 37° C. and developed. Control sections were overlaid with a milk agarmixture without plasminogen. Other controls included those in whicheither 100 μg/ml anti-tPA (a generous gift of T. Podor, MacMasterUniversity), or anti-uPA (Chemicon International, Temecula, Calif.)antibodies or 5 μM BAIT were included in addition to plasminogen.

[0475] Immunohistochemistry:

[0476] All immunohistochemistry was performed on 5 μmdeparaffinized-embedded sections. The sections were first immersed inmethanol 0.3% H₂O₂ for 30 minutes and then either preincubated directlywith 10% serum (either horse or goat), or first treated with 0.04%pepsin in 0.1N HCl for 20 minutes at 23° C. prior to being blocked withserum. All sections were also developed with the ABC reagent (VectorLaboratories, Burlingame, Calif.), using the DAB chromogen for 4 min,after which the sections were counter-stained with Mayer's hematoloxylinfor 1 minute. For BAIT staining, adult male Sprague-Dawley rats thatwere not injected with BAIT or PBS, were sacrificed 6, 24, 48, 72, 96,or 168 hours after bipolar coagulation of the middle cerebral artery, orsham operation, and sections were prepared as above and stained withrabbit anti-human BAIT as described. Pulsinelli A., et. al., in BarnettHJM et al (ed): Stroke. Pathophysiology, Diagnosis and Management. NewYork, Churchill Livingstone; 1992:49-68. For tPA, uPA and laminin, bothcontrol and BAIT treated animals, were examined. For tPA the sectionswere stained with affinity purified sheep anti-human tPA (a generousgift from Tom Podor, MacMaster University), at 1:800 dilution afterpepsin digestion. For uPA goat anti-human uPA (ChemiconInternational-AB767, Temecula, Calif.) was used at 1:200 dilution afterpepsin digestion. For laminin staining a murine monoclonal anti-humanlaminin (Chemicon International-MAB2920, Temecula, Calif.) was used at a1:4000 dilution either with or without pepsin digestion as above. Forall immunohistochemical analysis n≧2 for each condition at each timepoint except for BAIT staining at 96 and 168 hours for which n=l each.

[0477] 7(a)—BAIT Expression After Stroke:

[0478] Since tPA may contribute to neuronal death following cerebralinfarction, then increased expression of BAIT might play an importantrole in neuronal survival after stroke. To examine the expression ofBAIT following cerebral ischemia, immunohistochemical staining of brainsections was performed at 6, 24, 48, 72, 96 and 168 hours after middlecerebral artery occlusion and reperfusion. FIG. 6 shows threerepresentative brain sections harvested 72 hours after reperfusion andstained with hematoxilin-eosin. The infarct is clearly evident as thelighter stained tissue in the cortex of the left hemisphere, and the boxindicates the area where higher resolution analysis was performed. BAITimmunoreactivity was seen to be increased in the area surrounding theischemic core (penumbra) and in the ipsilateral hippocampus as early as6 hours after stroke, and remained elevated up to 168 hours whencompared with the contralateral, non ischemic, hemisphere or with shamoperated controls (data not shown). The peak of BAIT immunoreactivity inboth the number of BAIT positive cells, and in the intensity of thestaining, appeared to be at 48 hours following reperfusion (FIG. 7). Theapparent rapid increase in BAIT expression following infarction suggeststhat the surrounding surviving cells may be upregulating BAIT expressionin response to the ischemic insult.

[0479] 7(b)—Effect of BAIT on Stroke Volume.

[0480] To see if BAIT could reduce neuronal cell death after stroke withsubsequent preservation of normal brain tissue, BAIT was administeredintracerebrally immediately following MCA occlusion. Comparison ofstroke volume between control and BAIT treated animals 72 hours afterreperfusion indicated that intracortical injection of 30 μM BAIT reducedstroke size by 64%, from 161 mm³in control animals to 58 mm³ in BAITtreated animals (FIG. 8). In contrast, stroke volume in animals treatedwith inactive BAIT, cleaved in its reactive center loop, showed nodecrease in stroke size relative to control animals, suggesting thatactive BAIT is required to reduce stroke volume (FIG. 8).

[0481] 7(c)—Proteinase Activity After Stroke:

[0482] Since only the active inhibitory form of BAIT reduced strokevolume, this suggests that BAIT acts primarily by blocking proteinaseactivity, possibly tPA activity. To examine proteinase activityfollowing stroke, and to determine the effect of BAIT treatment onproteinase activity, two different assays were utilized. The first,SDS-PAGE zymography, was performed on extracts of tissues dissected fromthe cortex, either ipsi- or contralateral to the stroke of both PBS- andBAIT-treated animals (FIG. 9A). Following electrophoresis and removal ofSDS, the gels were overlaid onto milk-agarose gels with or withoutplasminogen. In the absence of plasminogen no proteinase activity couldbe detected in any of the extracts, whereas addition of plasminogen tothe milk-agarose mixture demonstrated that both tPA and uPA activitywere present in all cortex extracts examined, including those from shamoperated animals (data not shown). Examination of extracts prepared fromanimals 6 hours following reperfusion suggested that both tPA and uPAactivity were elevated ipsilateral to the stroke in PBS-treated animals,but that only uPA appeared to be elevated in BAIT-treated brains (FIG.9A). However, by 72 hours tPA activity appeared to return to baseline,indicating that the increase in tPA activity is transient and that BAITcan reduce the extent of this increase. In contrast, uPA-catalyzedactivity, which was relatively low in the 6-hour extracts, increaseddramatically in ipsilateral extracts of animals sacrificed 72 hoursafter reperfusion, and this increase was apparent in both control andBAIT-treated brains. However, like both tPA and uPA at 6 hours, theamount of uPA activity at 72 hours was significantly lower inBAIT-treated animals compared to controls (FIG. 9A). Quantitative imageanalysis of these data indicated that by 6 hour following reperfusionipsilateral to the stroke in PBS-treated animals there was anapproximately 50% increase in tPA activity and an approximately 125%increase in uPA activity relative to baseline levels (FIG. 9B). However,in BAIT-treated animals the increase of both PAs ipsilateral to thestroke was markedly reduced, showing only an approximately 50% increasefor uPA and no significant increase in tPA compared to baseline levels(FIG. 9B). These results indicate that there is an early and transientincrease in tPA activity ipsilateral to the stroke, and that BAIT isable to block this increase. Similarly, there is an early increase inuPA activity ipsilateral to the stroke, but in contrast to tPA thisincrease is not transient and continues to rise at least up to 72 hoursafter reperfusion, and is not blocked by treatment with BAIT but is onlyreduced compared to the PBS-treated animals.

[0483] To examine the distribution of proteinase activity within thebrains of the PBS- and BAIT-treated animals, in situ zymography offrozen brain sections was performed. These data demonstrate that likethe SDS-PAGE zymography, all of the proteolytic activity detected inboth control and BAIT treated brains was plasminogen-dependent, since noproteinase activity was observed in the absence of plasminogen (FIG. 10,panels A & D). At 6 hours following reperfusion, proteinase activity inall sections was primarily associated with the meningeal tissues of bothipsi- and contralateral sides. This activity was also completely blockedby the addition of anti-tPA antibodies indicating that the majority ofPA activity within the brain at this time is tPA (data not shown). Incontrast, by 72 hours following reperfusion, there was a large increasein plasminogen-dependent proteolytic activity ipsilateral to the strokein control animals (FIG. 10B), and unlike the 6 hour sections or the 72hour contralateral side, this activity was not restricted to themeninges and was not completely blocked by the addition of anti-tPAantibodies to the plasminogen overlay (arrows in FIGS. 10B-C). InBAIT-treated animals this zone of proteinase activity was significantlysmaller than in the untreated animals (FIGS. 10E-F). This suggests thatby 72 hours much of the plasminogen dependent activity within the regionof the stroke was not tPA. Consistent with this, the addition ofanti-uPA antibodies to the plasminogen overlay markedly reducedproteolysis within the area of the stroke while having no effect on theproteolytic activity in the meningeal tissues contralateral to thestroke (data not shown). This implies that within the area of theinfarct at 72 hour following reperfusion there is a significant increasein uPA activity. These results also suggest that there is not a largeup-regulation of either tPA or uPA immediately following stroke,however, by 72 hours after reperfusion, uPA-catalyzed proteolysis issignificantly increased specifically within the region of the infarct.These results are also consistent with the SDS-PAGE zymography. andsuggest that the lesser increase in uPA activity observed by SDS-PAGEzymography in BAIT-treated animals, may simply reflect the smaller sizeof the infarct in this group and not a direct inhibition of theup-regulation of uPA-activity by BAIT.

[0484] Immunohistochemical staining for tPA indicated that by 6 hoursfollowing reperfusion, tPA antigen was detected only within the vascularendothelial cells and not within neuronal cells (FIG. 10G). Consistentwith the relatively low levels of uPA activity at 6 hours, no uPAstaining could be detected in these sections (data not shown). However,by 72 hours after reperfusion, uPA immunoreactivity was readilydetected, but only in the area of ischemic penumbra (FIG. 10H). This isconsistent with the in situ zymography analysis demonstrating uPAactivity predominantly within the cortex and only at 72 hours afterreperfusion. Finally, at 72 hours in BAIT-treated animals, there was amarked reduction in the overall area where uPA antigen was detected butnot in the intensity of the staining, compared to PBS-treated animals(data not shown). This further suggests that the reduced uPA activityobserved by zymography was likely due to the reduced size of the infarctin BAIT-treated animals.

[0485] Basement Membrane Degradation After Cerebral Ischemia:

[0486] Since excitotoxin-induced laminin degradation has been suggestedto be mediated by tPA and to precede apoptotic cell death, we examinedthe effect of stroke on laminin immunoreactivity. For this analysis weutilized a monoclonal antibody that does not react strongly with ratlaminin in fixed tissue unless the tissue is first proteolyzed to exposecryptic laminin epitopes. This is shown in FIG. 11 panels A and B, wherepanel A shows a section of un-proteolyzed rat cortex reacted with theantibody, and panel B shows an adjacent section that was first treatedwith proteinase in vitro before reaction with the antibody. Theseresults indicate that in the absence of proteolysis this antibody doesnot react with vascular laminin. However, after proteolysis there is astrong reaction that appears to be localized to the vessels. Thus thisantibody provides an excellent tool to probe for partial proteolysis ofthe basement membrane within fixed brain tissue. Examination of lamininstaining in cortical tissue as early as 10 min. after reperfusionindicated that even at this early time there was apparently significantproteolysis of the basement membrane in control animals (FIG. 11C).However, in BAIT treated animals the extent of laminin proteolysis wassignificantly reduced such that only slight staining of the vascularlaminin was apparent (FIG. 11D). This latter result was not due to theabsence of laminin in this tissue since treatment of the sections withproteinase in vitro yielded staining indistinguishable from that shownin FIG. 11B (data not shown). Vascular laminin staining was alsoobserved at 6 hours after reperfusion in control animals and, similar tothe results at 10 minutes, treatment with BAIT significantly reducedthis staining (FIGS. 11E-F). Furthermore, by 6 hours after reperfusionlaminin staining was also observed within neurons in the area ofcerebral ischemia and, as with vascular laminin staining, was reduced byBAIT treatment (FIGS. 11E-F). The neuronal staining most likelyrepresents new synthesis of laminin since in control animals notsubjected to stroke no laminin staining was observed in neurons eitherwith or without proteinase treatment (FIGS. 11A-B). Laminin stainingremained strong at 24 and 48, but started to decrease by 72 hours. Also,at each time point the BAIT treated animals showed significantly lessimmuno-reactivity than control animals (data not shown). These datasuggest that there is a very early proteolytic event that appears to acton the vascular basement membrane, and that BAIT treatment is able toreduce this proteolysis.

[0487] Apoptosis:

[0488] Since cerebral ischemia has been suggested to induce apoptosis inthe ischemic penumbra, then a good therapeutic strategy aimed atreducing cell death after stroke should target the recovery of cells inthis area. To see if BAIT reduced infarct volume by preventing penumbralapoptosis, tissue from untreated and BAIT treated animals was stained bythe TUNEL method (FIGS. 12A-C). The extent of apoptosis in thesesections was then quantified as described above and these data are shownin FIG. 12D. The number of cells within a defined area of the penumbrawith apoptotic bodies after 72 hours of cerebral ischemia was 22±5 inuntreated animals and decreased to 8±2 in BAIT-treated animals (FIG.12D). This indicates that BAIT significantly inhibits penumbralapoptosis. To see if BAIT also blocked cell death at earlier times,apoptosis was also quantified at 6, 24, and 48 hours. These dataindicate that at all times examined apoptosis was reduced by at least50% with BAIT treatment (FIG. 12E). Finally, in order to test if BAIThad a direct effect on apoptosis, two independent assays were performed.In the first, BAIT was tested for its ability to block T-cell receptormediated apoptosis of a T-cell hybridoma in vitro. In the second assayBAIT was tested for its ability to directly inhibit caspase activity inextracts of B lymphoma cells treated with anti-Fas IgG to induceapoptosis and caspase activation. In both assays BAIT had no effect oneither apoptosis or caspase activity (data not shown). Taken together,these results indicate that BAIT is not a direct inhibitor of apoptosis,and therefore, it is likely that BAIT blocks events prior to inductionof apoptosis.

[0489] Discussion.

[0490] BAIT, a natural inhibitor of tPA, is found almost exclusivelywithin the central nervous system, and shows an early and significantincrease in its expression within the area of ischemic penumbra inresponse to stroke (FIG. 7). Cerebral ischemia is known to induceneuronal depolarization, as well as release of excitotoxins, which inturn trigger the release of tPA. Since tPA may be associated withincreased neuronal loss in response to both ischemia and excitotoxins,then the increased local expression of BAIT following ischemia mayrepresent an innate protective response to elevated tPA levels, andsuggests that BAIT may be a naturally occurring neuronal survivalfactor. Consistent with this hypothesis, BAIT-treatment resulted in asignificant decrease in stroke volume relative to control animals.Furthermore, only functionally active BAIT was able to reduce infarctsize, suggesting that inhibition of proteinase activity was necessaryfor BAIT's neuroprotective effects (FIG. 8).

[0491] Zymographic analysis of brain extracts at 6 and 72 hours afterreperfusion indicated that there was an early rise in both tPA and uPAactivity in the area of the infarct in control animals, and thattreatment with BAIT significantly reduced these activities (FIG. 9).These data are similar to earlier results that reported an increase inuPA activity in both rats and mice following cerebral ischemia, and toat least one other study that reported a significant increase in tPAactivity. However, in two studies tPA activity following stroke wasreported to be either decreased, or unchanged. The apparent differencein the activity of tPA noted here compared to these earlier studies mayin part reflect the time after cerebral ischemia when tPA was measuredsince our data suggest that the increase in tPA activity is transientand since none of these other studies measured tPA at six hoursfollowing reperfusion. These differences might also be due to thedifferent animal models used in the various studies. For example, themodel used here creates a permanent occlusion of the middle cerebralartery at its crossing point with the inferior cerebral vein withreperfusion provided by temporary clamping of the left carotid artery.This produces an ischemic injury in a very well defined area of thecerebral cortex (FIG. 6), and in contrast to the intravascular filamentmodel (Longa E Z, et. al., Stroke 1989;20:84-91), avoids the potentialof large lesions to the vascular endothelium and severe disruption ofthe blood-brain barrier that could lead to significant changes in thelocal tPA activity. Nonetheless, our results and those of Wang et al(Wang Y F, et. al. Nat Med. 1998;4:228-231) suggest that there is anearly local increase in tPA activity in the area of the infarct, and thedata reported here further suggest that this increase is transient.Since treatment with functionally active BAIT reduced both the localincrease in tPA activity as well as the infarct size, it is possiblethat these two effects are related, and that by blocking the action oftPA very early after reperfusion the later increase of the infarctedarea is prevented.

[0492] In contrast to tPA, uPA activity increased to very high levels by72 hours following reperfusion, and was localized almost exclusively tothe ischemic penumbra (FIGS. 9 & 10). The role of uPA after cerebralischemia is largely unknown. However, since the necrotic core is alreadywell defined by 72 hours after the stroke, it is unlikely that the lateincrease in uPA activity plays an important role in the development ofthe infarct. This inference is also consistent with a recent study ofstroke in uPA deficient mice that indicated that there was no differencein infarct volume between wild-type and uPA−/− mice 24 hours afterreperfusion. However, since uPA has been demonstrated in both glialcells during myelination and in mature cortical neurons (Del Bigio M R,et. al., Brain Res.Dev.Brain Res. 1995;86:345-347), the late expressionof uPA activity and antigen suggests that uPA could participate in theprocess of neuronal recovery after stroke as was suggested by Rosenberget al. (Rosenberg G A, et. al., J Cereb.Blood Flow Metab.1996;16:360-366).

[0493] Although the role of tPA activity in infarct evolution is notwell understood, tPA induced plasmin cleavage of basement membranelaminin has been suggested to play a role in excitotoxin inducedneuronal death within the hippocampus and in the disappearance ofbasement membrane antigens following ischemia and reperfusion. Thebasement membrane is a specialized part of the extracellular matrix thatconnects the endothelial cell compartment to the surrounding celllayers. Laminins are very important components of the basement membrane,playing a pivotal role in cell- extracellular matrix interactions,including promotion of neurite outgrowth, cell attachment,proliferation, and differentiation, as well as in the development, andregeneration of the nervous system. Paulsson M,Crit.Rev.Biochem.Mol.Biol. 1992;27:93-127; Calof A L, et. al., Neuron1994;13:117-130; Hammarback J A, et. al., Dev.Biol. 1988;126:29-39;Liesi P, EMBO J 1985;4:2505-2511; Millaruelo A I, et. al., Brain Res.1988;466:219-228. In the present study exposure of cryptic lamininepitopes within the basement membrane was observed within 10 minutes ofreperfusion, suggesting that there is proteolytic activity acting on thebasement membrane very early following cerebral ischemia (FIG. 11). Likethe observed increase in tPA, this activity appears to be transient withpeak epitope exposure occurring within 6-24 hours of reperfusion.Whether this effect is due to the direct action of tPA on laminin, ismediated through plasmin, or involves MMPs or other as yet unidentifiedproteinases is not clear. Regardless of which proteinase is responsiblefor the apparent basement membrane degradation, the extent of lamininepitope expression was significantly decreased in BAIT-treated animals(FIG. 11). This suggests that BAIT is inhibiting the proteolytic attackon the basement membrane, most likely by inhibiting tPA. Thus, BAIT, byblocking the early increase in tPA activity, may be able to preserve theintegrity of the basement membrane and thus the blood brain barrierafter stroke.

[0494] It is known that disruption of cell-matrix interactions can leadto apoptosis. Meredith J E J, et. al. Mol.Biol.Cell 1993;4:953-961;Murtomaki S, et. al., Dev.Biol. 1995;168:635-648. Since the crypticlaminin epitopes were observed as early as 10 minutes after reperfusion,with high levels of neuronal expression seen by 6 hours (FIG. 11), wellbefore 24-48 hours, the peak of apoptosis (FIG. 12E), this suggests thatthe proteolytic disruption of the basement membrane may be the triggerthat initiates the program of apoptotic neuronal cell death. Thus, thecapacity of active BAIT to block tPA-induced degradation of the basementmembrane may explain the ability of BAIT treatment to reduce neuronalapoptosis by nearly 70% (FIG. 12D). Finally, although it has beendemonstrated that apoptotic cell death in stroke is mediated byproteinases known as caspases (Barinaga M, Science 1998;280:32-34; ChengY, et. al., J Clin.Invest. 1998;101:1992-1999; Namura S, et. al., JNeurosci. 1998;18:3659-3668), BAIT failed to inhibit either caspaseactivity or T-cell apoptosis, suggesting that BAIT is not an inhibitorof apoptosis per se and does not directly block caspase activation oractivity.

[0495] Intra-neuronal laminin-like immunoreactivity has been reported inboth the developing and adult central nervous system, and in astrocytesafter transient ischemia. Suzuki H, et. al., Brain Res.1990;520:324-329; Jucker M, et. al., Ann.N.Y.Acad.Sci.1993;679:245-52:245-252. Laminin has also been shown to promote neuriteoutgrowth. We observed morphologically healthy neurons that werepositive for intracellular laminin staining in the area of penumbra incontrol animals by 6 hours after reperfusion (FIG. 11E), suggesting arole for intraneuronal laminin in neuronal maintenance followingischemia. It is possible that synthesis of laminin by neurons is inresponse to laminin degradation within the basement membrane, and thatBAIT treated animals show fewer laminin positive cells because there isless basement membrane degradation and thus many fewer distressed cells.It is interesting to note that the region of the cortex that shows manylaminin positive cells at six hours after reperfusion is the same regionthat shows many apoptotic cells at 48 hours. This suggests that iflaminin expression is an early marker for cell distress then BAITtreatment must be acting very early in the pathway that leads toneuronal apoptosis.

[0496] Taken together the data presented here suggest a model for strokeinduced neuronal death within the ischemic penumbra, and demonstrate thepotential therapeutic benefits of BAIT in this setting. We speculatethat one of the first potentially deleterious events to occur is therelease of tPA by the vascular endothelial cells in response to theacute ischemia. If there is also increased vascular permeability at thistime as a result of damage to the blood-brain barrier, then this willallow tPA to cross from the lumen of the vessel into to the subvascularspace, where it can bind directly to laminin. This inappropriatelytargeted tPA can then, either by itself or in concert with otherproteinases such as plasmin, or MMPs begin to degrade the basementmembrane. This leads to a further increase in vascular permeability,which in turn may accelerate the degradation of the blood brain barrier.In addition, neuronal cells that are also dependant upon the integrityof the basement membrane may begin to loose their contacts with thesubstrate, which in itself might induce a program of apoptosis as hasbeen described for other cell types. Our data also suggest that byapproximately 72 hours after the stroke onset the basement membranedegradation and neuronal apoptosis have decreased, stabilizing thelesion. At this time other factors such as uPA are up-regulated possiblyas part of the recovery process. BAIT treatment, by blocking the earlyeffects of proteinase activity, may help to maintain the integrity ofthe basement membrane, preventing further passage of tPA or otherpotentially harmful blood born factors to the subvascular space. BAITmay also directly prevent neuronal loss by helping to preserve neuronalcontacts to the basement membrane.

[0497] Moreover, BAIT polypeptides (including N & C terminal mutants,variants, and antibodies described herein) and polynucleotides may alsobe used to treat cerebrovascular disorders, such as carotid arterydisease, cerebral amyloid angiopathy, cerebral aneurysm, cerebralanoxia, cerebral arteriosclerosis, cerebral arteriosclerosis, cerebralarteriovenous malformations, cerebral artery disease, cerebral embolism,cerebral thrombosis, cerebral hemorrhage (e.g., hematoma), cerebralinfarction, cerebral ischemia (e.g., transient cerebral ischemia,subclavian steal syndrome, and vertebrobasilar insufficiency), vasculardementia, leukomalacia, vascular headache (e.g., cluster headache,migraine), and/or strokes.

[0498] Additionally, BAIT polypeptides (including N & C terminalmutants, variants, and antibodies) and polynucleotides describedherein)may be used to inhibit tPA and/or increase uPA. Also, BAITpolypeptides (including N & C terminal mutants, variants, andantibodies) and polynucleotides may be used to activate tPA and/ordeacrease uPA.

[0499] Furthermore, polypeptides (including N & C terminal mutants,variants, and antibodies) and polynucleotides may be used to treat braindiseases caused by a variety etiologies. For example, polypeptides(including N & C terminal mutants, variants, and antibodies) andpolynucleotides may be used to treat akinetic mutism, auditory diseases,basal ganglia diseases (e.g., Huntington's Disease, Parkinson Disease,or Alzheimer's Disease), brain abscess, chronic brain damage (e.g.,cerebral palsy), metabolic brain diseases (e.g., abetalipoproteinemia,PKU, Lesch-Nyan Syndrome), brain edema, and brain neoplasms. Similarly,BAIT may be used to treat cerebellar diseases (e.g., ataxia), andcerebral sclerosis, dementia, encephalitis, encephalomalacia, epilepsy,Hallervorden-Spatz Syndrome, hydrocephalus, hypothalamic disease,malaria, narcolepsy, poliomyelitis, pseudotumor cerebri, Rett Syndrome,Reye's Syndrome, Thalamic Disease, Toxoplasmosis, IntracranialTuberculoma, and Zellweger Syndrome. Thus, diseases of the brain and thenervous system may be treatable using polypeptides (including N & Cterminal mutants, variants, and antibodies) and polynucleotides.

Example 8 Production of an Antibody

[0500] a) Hybridoma Technology

[0501] The antibodies of the present invention can be prepared by avariety of methods. (See, Current Protocols, Chapter 2.) As one exampleof such methods, cells expressing polypeptide(s) of the invention areadministered to an animal to induce the production of sera containingpolyclonal antibodies. In a preferred method, a preparation ofpolypeptide(s) of the invention is prepared and purified to render itsubstantially free of natural contaminants. Such a preparation is thenintroduced into an animal in order to produce polyclonal antisera ofgreater specific activity.

[0502] Monoclonal antibodies specific for polypeptide(s) of theinvention are prepared using hybridoma technology. (Kohler et al.,Nature 256:495 (1975); Kohler et al., Eur. J. Immunol. 6:511 (1976);Kohler et al., Eur. J. Immunol. 6:292 (1976); Hammerling et al., in:Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-681(1981)). In general, an animal (preferably a mouse) is immunized withpolypeptide(s) of the invention or, more preferably, with a secretedpolypeptide-expressing cell. Such polypeptide-expressing cells arecultured in any suitable tissue culture medium, preferably in Earle'smodified Eagle's medium supplemented with 10% fetal bovine serum(inactivated at about 56° C.), and supplemented with about 10 g/l ofnonessential amino acids, about 1,000 U/ml of penicillin, and about 100μg/ml of streptomycin.

[0503] The splenocytes of such mice are extracted and fused with asuitable myeloma cell line. Any suitable myeloma cell line may beemployed in accordance with the present invention; however, it ispreferable to employ the parent myeloma cell line (SP20), available fromthe ATCC. After fusion, the resulting hybridoma cells are selectivelymaintained in HAT medium, and then cloned by limiting dilution asdescribed by Wands et al. (Gastroenterology 80:225-232 (1981)). Thehybridoma cells obtained through such a selection are then assayed toidentify clones which secrete antibodies capable of binding thepolypeptide(s) of the invention.

[0504] Alternatively, additional antibodies capable of binding topolypeptide(s) of the invention can be produced in a two-step procedureusing anti-idiotypic antibodies. Such a method makes use of the factthat antibodies are themselves antigens, and therefore, it is possibleto obtain an antibody which binds to a second antibody. In accordancewith this method, protein specific antibodies are used to immunize ananimal, preferably a mouse. The splenocytes of such an animal are thenused to produce hybridoma cells, and the hybridoma cells are screened toidentify clones which produce an antibody whose ability to bind to theprotein-specific antibody can be blocked by polypeptide(s) of theinvention. Such antibodies comprise anti-idiotypic antibodies to theprotein-specific antibody and are used to immunize an animal to induceformation of further protein-specific antibodies.

[0505] For in vivo use of antibodies in humans, an antibody is“humanized”. Such antibodies can be produced using genetic constructsderived from hybridoma cells producing the monoclonal antibodiesdescribed above. Methods for producing chimeric and humanized antibodiesare known in the art and are discussed herein. (See, for review,Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214(1986); Cabilly et al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP171496; Morrison et al., EP 173494; Neuberger et al., WO 8601533;Robinson et al., WO 8702671; Boulianne et al., Nature 312:643 (1984);Neuberger et al., Nature 314:268 (1985).)

[0506] b) Isolation of Antibody Fragments Directed AgainstPolypeptide(s) From a Library of scFvs

[0507] Naturally occurring V-genes isolated from human PBLs areconstructed into a library of antibody fragments which containreactivities against polypeptide(s) of the invention to which the donormay or may not have been exposed (see e.g., U.S. Pat. 5,885,793incorporated herein by reference in its entirety).

[0508] Rescue of the Library.

[0509] A library of scFvs is constructed from the RNA of human PBLs asdescribed in PCT publication WO 92/01047. To rescue phage displayingantibody fragments, approximately 109 E. coli harboring the phagemid areused to inoculate 50 ml of 2xTY containing 1% glucose and 100 μg/ml ofampicillin (2xTY-AMP-GLU) and grown to an O.D. of 0.8 with shaking. Fiveml of this culture is used to innoculate 50 ml of 2xTY-AMP-GLU, 2×108 TUof delta gene 3 helper (M13 delta gene III, see PCT publication WO92/01047) are added and the culture incubated at 37° C. for 45 minuteswithout shaking and then at 37° C. for 45 minutes with shaking. Theculture is centrifuged at 4000 r.p.m. for 10 min. and the pelletresuspended in 2 liters of 2xTY containing 100 μg/ml ampicillin and 50ug/ml kanamycin and grown overnight. Phage are prepared as described inPCT publication WO 92/01047.

[0510] M13 delta gene III is prepared as follows: M13 delta gene IIIhelper phage does not encode gene III protein, hence the phage(mid)displaying antibody fragments have a greater avidity of binding toantigen. Infectious M13 delta gene III particles are made by growing thehelper phage in cells harboring a pUC19 derivative supplying the wildtype gene III protein during phage morphogenesis. The culture isincubated for 1 hour at 37° C. without shaking and then for a furtherhour at 37° C. with shaking. Cells are spun down (IEC-Centra 8,400r.p.m. for 10 min), resuspended in 300 ml 2xTY broth containing 100 μgampicillin/ml and 25 μg kanamycin/ml (2xTY-AMP-KAN) and grown overnight,shaking at 37° C. Phage particles are purified and concentrated from theculture medium by two PEG-precipitations (Sambrook et al., 1990),resuspended in 2 ml PBS and passed through a 0.45 μm filter (MinisartNML; Sartorius) to give a final concentration of approximately 1013transducing units/ml (ampicillin-resistant clones).

[0511] Panning of the Library.

[0512] Immunotubes (Nunc) are coated overnight in PBS with 4 ml ofeither 100 μg/ml or 10 μg/ml of a polypeptide of the present invention.Tubes are blocked with 2% Marvel-PBS for 2 hours at 37° C. and thenwashed 3 times in PBS. Approximately 1013 TU of phage is applied to thetube and incubated for 30 minutes at room temperature tumbling on anover and under turntable and then left to stand for another 1.5 hours.Tubes are washed 10 times with PBS 0.1% Tween-20 and 10 times with PBS.Phage are eluted by adding 1 ml of 100 mM triethylamine and rotating 15minutes on an under and over turntable after which the solution isimmediately neutralized with 0.5 ml of 1.0 M Tris-HCl, pH 7.4. Phage arethen used to infect 10 ml of mid-log E. coli TG1 by incubating elutedphage with bacteria for 30 minutes at 37° C. The E. coli are then platedon TYE plates containing 1% glucose and 100 μg/ml ampicillin. Theresulting bacterial library is then rescued with delta gene 3 helperphage as described above to prepare phage for a subsequent round ofselection. This process is then repeated for a total of 4 rounds ofaffinity purification with tube- washing increased to 20 times with PBS,0.1% Tween-20 and 20 times with PBS for rounds 3 and 4.

[0513] Characterization of Binders.

[0514] Eluted phage from the 3rd and 4th rounds of selection are used toinfect E. coli HB 2151 and soluble scFv is produced (Marks, et al.,1991) from single colonies for assay. ELISAs are performed withmicrotitre plates coated with either 10 pg/ml of the polypeptide of thepresent invention in 50 mM bicarbonate pH 9.6. Clones positive in ELISAare further characterized by PCR fingerprinting (see, e.g., PCTpublication WO 92/01047) and then by sequencing. These ELISA positiveclones may also be further characterized by techniques known in the art,such as, for example, epitope mapping, binding affinity, receptor signaltransduction, ability to block or competitively inhibit antibody/antigenbinding, and competitive agonistic or antagonistic activity.

Example 9 Protein Fusions

[0515] The polypeptides of the present invention are preferably fused toother proteins. These fusion proteins can be used for a variety ofapplications. For example, fusion of the present polypeptides toHis-tag, HA-tag, protein A, IgG domains, and maltose binding proteinfacilitates purification. (See Examples above; see also EP A 394,827;Traunecker, et al., Nature 331:84-86 (1988).) Similarly, fusion toIgG-1, IgG-3, and albumin increases the halflife time in vivo. Nuclearlocalization signals fused to the polypeptides of the present inventioncan target the protein to a specific subcellular localization, whilecovalent heterodimer or homodimers can increase or decrease the activityof a fusion protein. Fusion proteins can also create chimeric moleculeshaving more than one function. Finally, fusion proteins can increasesolubility and/or stability of the fused protein compared to thenon-fused protein. All of the types of fusion proteins described abovecan be made by modifying the following protocol, which outlines thefusion of a polypeptide to an IgG molecule, or the protocol described inthe Examples.

[0516] Briefly, the human Fc portion of the IgG molecule can be PCRamplified, using primers that span the 5′ and 3′ ends of the sequencedescribed below. These primers also should have convenient restrictionenzyme sites that will facilitate cloning into an expression vector,preferably a mammalian expression vector.

[0517] For example, if pC4 (Accession No. 209646) is used, the human Fcportion can be ligated into the BamHI cloning site. Note that the 3′BamHI site should be destroyed. Next, the vector containing the human Fcportion is re-restricted with BamHI, linearizing the vector, and apolynucleotide of the present invention, isolated by the PCR protocoldescribed in Example 1, is ligated into this BarnHI site. Note that thepolynucleotide is cloned without a stop codon, otherwise a fusionprotein will not be produced.

[0518] If the naturally occurring signal sequence is used to produce thesecreted protein, pC4 does not need a second signal peptide.Alternatively, if the naturally occurring signal sequence is not used,the vector can be modified to include a heterologous signal sequence.(See, e.g., WO 96/34891.)

[0519] Human IgG Fc Region:      GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAATT(SEQ ID NO: )CGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGGTGGTGGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGCCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAACCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTGCGACGGCCGCGACTCTAGAGGAT

Example 10 Method of Detecting Abnormal Levels of a Polypeptide in aBiological Sample

[0520] A polypeptide of the present invention can be detected in abiological sample, and if an increased or decreased level of thepolypeptide is detected, this polypeptide is a marker for a particularphenotype. Methods of detection are numerous, and thus, it is understoodthat one skilled in the art can modify the following assay to fit theirparticular needs.

[0521] For example, antibody-sandwich ELISAs are used to detectpolypeptides in a sample, preferably a biological sample. Wells of amicrotiter plate are coated with specific antibodies, at a finalconcentration of 0.2 to 10 ug/ml. The antibodies are either monoclonalor polyclonal and are produced by the method described in the Examples.The wells are blocked so that non-specific binding of the polypeptide tothe well is reduced.

[0522] The coated wells are then incubated for >2 hours at RT with asample containing the polypeptide. Preferably, serial dilutions of thesample should be used to validate results. The plates are then washedthree times with deionized or distilled water to remove unboundedpolypeptide.

[0523] Next, 50 ul of specific antibody-alkaline phosphatase conjugate,at a concentration of 25-400 ng, is added and incubated for 2 hours atroom temperature. The plates are again washed three times with deionizedor distilled water to remove unbounded conjugate.

[0524] Add 75 ul of 4-methylumbelliferyl phosphate (MUP) orp-nitrophenyl phosphate (NPP) substrate solution to each well andincubate 1 hour at room temperature. Measure the reaction by amicrotiter plate reader. Prepare a standard curve, using serialdilutions of a control sample, and plot polypeptide concentration on theX-axis (log scale) and fluorescence or absorbance of the Y-axis (linearscale). Interpolate the concentration of the polypeptide in the sampleusing the standard curve.

Example 11 Formulation

[0525] The invention also provides methods of treatment and/orprevention diseases, disorders, and/or conditions (such as, for example,any one or more of the diseases or disorders disclosed herein) byadministration to a subject of an effective amount of a Therapeutic. Bytherapeutic is meant a polynucleotides or polypeptides of the invention(including fragments and variants), agonists or antagonists thereof,and/or antibodies thereto, in combination with a pharmaceuticallyacceptable carrier type (e.g., a sterile carrier).

[0526] The Therapeutic will be formulated and dosed in a fashionconsistent with good medical practice, taking into account the clinicalcondition of the individual patient (especially the side effects oftreatment with the Therapeutic alone), the site of delivery, the methodof administration, the scheduling of administration, and other factorsknown to practitioners. The “effective amount” for purposes herein isthus determined by such considerations.

[0527] As a general proposition, the total pharmaceutically effectiveamount of the Therapeutic administered parenterally per dose will be inthe range of about 1 ug/kg/day to 10 mg/kg/day of patient body weight,although, as noted above, this will be subject to therapeuticdiscretion. More preferably, this dose is at least 0.01 mg/kg/day, andmost preferably for humans between about 0.01 and 1 mg/kg/day for thehormone. If given continuously, the Therapeutic is typicallyadministered at a dose rate of about 1 ug/kg/hour to about 50ug/kg/hour, either by 1-4 injections per day or by continuoussubcutaneous infusions, for example, using a mini-pump. An intravenousbag solution may also be employed. The length of treatment needed toobserve changes and the interval following treatment for responses tooccur appears to vary depending on the desired effect.

[0528] Therapeutics can be are administered orally, rectally,parenterally, intracistemally, intravaginally, intraperitoneally,topically (as by powders, ointments, gels, drops or transdermal patch),bucally, or as an oral or nasal spray. “Pharmaceutically acceptablecarrier” refers to a non-toxic solid, semisolid or liquid filler,diluent, encapsulating material or formulation auxiliary of any. Theterm “parenteral” as used herein refers to modes of administration whichinclude intravenous, intramuscular, intraperitoneal, intrasternal,subcutaneous and intraarticular injection and infusion.

[0529] Therapeutics of the invention are also suitably administered bysustained- release systems. Suitable examples of sustained-releaseTherapeutics are administered orally, rectally, parenterally,intracistemally, intravaginally, intraperitoneally, topically (as bypowders, ointments, gels, drops or transdermal patch), bucally, or as anoral or nasal spray. “Pharmaceutically acceptable carrier” refers to anon-toxic solid, semisolid or liquid filler, diluent, encapsulatingmaterial or formulation auxiliary of any type. The term “parenteral” asused herein refers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrasternal, subcutaneous andintraarticular injection and infusion.

[0530] Therapeutics of the invention are also suitably administered bysustained- release systems. Suitable examples of sustained-releaseTherapeutics include suitable polymeric materials (such as, for example,semi-permeable polymer matrices in the form of shaped articles, e.g.,films, or mirocapsules), suitable hydrophobic materials (for example asan emulsion in an acceptable oil) or ion exchange resins, and sparinglysoluble derivatives (such as, for example, a sparingly soluble salt).

[0531] Sustained-release matrices include polylactides (U.S. Pat. No.3,773,919, EP 58,481), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman et al., Biopolymers 22:547-556 (1983)),poly (2- hydroxyethyl methacrylate) (Langer et al., J. Biomed. Mater.Res. 15:167-277 (1981), and Langer, Chem. Tech. 12:98-105 (1982)),ethylene vinyl acetate (Langer et al., Id.) orpoly-D-(−)-3-hydroxybutyric acid (EP 133,988).

[0532] Sustained-release Therapeutics also include liposomally entrappedTherapeutics of the invention (see generally, Langer, Science249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy ofInfectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss,N.Y., pp. 317-327 and 353-365 (1989)). Liposomes containing theTherapeutic are prepared by methods known per se: DE 3,218,121; Epsteinet al., Proc. Nati. Acad. Sci. (USA) 82:3688-3692 (1985); Hwang et al.,Proc. Natl. Acad. Sci.(USA) 77:4030-4034 (1980); EP 52,322; EP 36,676;EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appl. 83-118008; U.S.Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily, theliposomes are of the small (about 200-800 Angstroms) unilamellar type inwhich the lipid content is greater than about 30 mol. percentcholesterol, the selected proportion being adjusted for the optimalTherapeutic.

[0533] In yet an additional embodiment, the Therapeutics of theinvention are delivered by way of a pump (see Langer, supra; Sefton, CRCCrit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507(1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)).

[0534] Other controlled release systems are discussed in the review byLanger (Science 249:1527-1533 (1990)).

[0535] For parenteral administration, in one embodiment, the Therapeuticis formulated generally by mixing it at the desired degree of purity, ina unit dosage injectable form (solution, suspension, or emulsion), witha pharmaceutically acceptable carrier, i.e., one that is non-toxic torecipients at the dosages and concentrations employed and is compatiblewith other ingredients of the formulation. For example, the formulationpreferably does not include oxidizing agents and other compounds thatare known to be deleterious to the Therapeutic.

[0536] Generally, the formulations are prepared by contacting theTherapeutic uniformly and intimately with liquid carriers or finelydivided solid carriers or both. Then, if necessary, the product isshaped into the desired formulation. Preferably the carrier is aparenteral carrier, more preferably a solution that is isotonic with theblood of the recipient. Examples of such carrier vehicles include water,saline, Ringer's solution, and dextrose solution. Non-aqueous vehiclessuch as fixed oils and ethyl oleate are also useful herein, as well asliposomes.

[0537] The carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, manose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium; and/or nonionicsurfactants such as polysorbates, poloxamers, or PEG.

[0538] The Therapeutic is typically formulated in such vehicles at aconcentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml, ata pH of about 3 to 8. It will be understood that the use of certain ofthe foregoing excipients, carriers, or stabilizers will result in theformation of polypeptide salts.

[0539] Any pharmaceutical used for therapeutic administration can besterile. Sterility is readily accomplished by filtration through sterilefiltration membranes (e.g., 0.2 micron membranes). Therapeuticsgenerally are placed into a container having a sterile access port, forexample, an intravenous solution bag or vial having a stopper pierceableby a hypodermic injection needle.

[0540] Therapeutics ordinarily will be stored in unit or multi-dosecontainers, for example, sealed ampoules or vials, as an aqueoussolution or as a lyophilized formulation for reconstitution. As anexample of a lyophilized formulation, 10-ml vials are filled with 5 mlof sterile-filtered 1% (w/v) aqueous Therapeutic solution, and theresulting mixture is lyophilized. The infusion solution is prepared byreconstituting the lyophilized Therapeutic using bacteriostaticWater-for-Injection.

[0541] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the Therapeutics of the invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition, theTherapeutics may be employed in conjunction with other therapeuticcompounds.

[0542] The Therapeutics of the invention may be administered alone or incombination with adjuvants. Adjuvants that may be administered with theTherapeutics of the invention include, but are not limited to, alum,alum plus deoxycholate (ImmunoAg), MTP-PE (Biocine Corp.), QS21(Genentech, Inc.), BCG, and MPL. In a specific embodiment, Therapeuticsof the invention are administered in combination with alum. In anotherspecific embodiment, Therapeutics of the invention are administered incombination with QS-21. Further adjuvants that may be administered withthe Therapeutics of the invention include, but are not limited to,Monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-21, QS-18,CRL1005, Aluminum salts, MF-59, and Virosomal adjuvant technology.Vaccines that may be administered with the Therapeutics of the inventioninclude, but are not limited to, vaccines directed toward protectionagainst MMR (measles, mumps, rubella), polio, varicella,tetanus/diptheria, hepatitis A, hepatitis B, haemophilus influenzae B,whooping cough, pneumonia, influenza, Lyme's Disease, rotavirus,cholera, yellow fever, Japanese encephalitis, poliomyelitis, rabies,typhoid fever, and pertussis. Combinations may be administered eitherconcomitantly, e.g., as an admixture, separately but simultaneously orconcurrently; or sequentially. This includes presentations in which thecombined agents are administered together as a therapeutic mixture, andalso procedures in which the combined agents are administered separatelybut simultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second.

[0543] The Therapeutics of the invention may be administered alone or incombination with other therapeutic agents. Therapeutic agents that maybe administered in combination with the Therapeutics of the invention,include but not limited to, other members of the TNF family,chemotherapeutic agents, antibiotics, steroidal and non- steroidalanti-inflammatories, conventional immunotherapeutic agents, cytokinesand/or growth factors. Combinations may be administered eitherconcomitantly, e.g., as an admixture, separately but simultaneously orconcurrently; or sequentially. This includes presentations in which thecombined agents are administered together as a therapeutic mixture, andalso procedures in which the combined agents are administered separatelybut simultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second.

[0544] In one embodiment, the Therapeutics of the invention areadministered in combination with members of the TNF family. TNF,TNF-related or TNF-like molecules that may be administered with theTherapeutics of the invention include, but are not limited to, solubleforms of TNF-alpha, lymphotoxin-alpha (LT-alpha, also known asTNF-beta), LT-beta (found in complex heterotrimer LT-alpha2-beta), OPGL,FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-gamma (InternationalPublication No. WO 96/14328), AIM-I (International Publication No. WO97/33899), endokine-alpha (International Publication No. WO 98/07880),TR6 (International Publication No. WO 98/30694), OPG, andneutrokine-alpha (International Publication No. WO 98/18921, OX40, andnerve growth factor (NGF), and soluble forms of Fas, CD30, CD27, CD40and 4-IBB, TR2 (International Publication No. WO 96/34095), DR3(International Publication No. WO 97/33904), DR4 (InternationalPublication No. WO 98132856), TR5 (International Publication No. WO98/30693), TR6 (International Publication No. WO 98/30694), TR7(International Publication No. WO 98/41629), TRANK, TR9 (InternationalPublication No. WO 98/56892), TR10 (International Publication No. WO98/54202), 312C2 (International Publication No. WO 98/06842), and TR12,and soluble forms CD154, CD70, and CD153.

[0545] In certain embodiments, Therapeutics of the invention areadministered in combination with antiretroviral agents, nucleosidereverse transcriptase inhibitors, non- nucleoside reverse transcriptaseinhibitors, and/or protease inhibitors. Nucleoside reverse transcriptaseinhibitors that may be administered in combination with the Therapeuticsof the invention, include, but are not limited to, RETROVIR™(zidovudine/AZT), VIDEX™ (didanosine/ddI), HIVID™ (zalcitabine/ddC),ZERIT™ io (stavudine/d4T), EPIVIR™ (lamivudine/3TC), and COMBIVIR™(zidovudine/lamivudine). Non-nucleoside reverse transcriptase inhibitorsthat may be administered in combination with the Therapeutics of theinvention, include, but are not limited to, VIRAMUNE™ (nevirapine),RESCRIPTOR™ (delavirdine), and SUSTIVA™ (efavirenz). Protease inhibitorsthat may be administered in combination with the Therapeutics of theinvention, include, but are not limited to, CRIXIVAN™ (indinavir),NORVIR™ (ritonavir), INVIRASE™ (saquinavir), and VIRACEPT™ (nelfinavir).In a specific embodiment, antiretroviral agents, nucleoside reversetranscriptase inhibitors, non-nucleoside reverse transcriptaseinhibitors, and/or protease inhibitors may be used in any combinationwith Therapeutics of the invention to treat AIDS and/or to prevent ortreat HIV infection.

[0546] In other embodiments, Therapeutics of the invention may beadministered in combination with anti-opportunistic infection agents.Anti-opportunistic agents that may be administered in combination withthe Therapeutics of the invention, include, but are not limited to,TRIMETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDLNE™, ATOVAQUONE™,ISONIAZID™, RIFAMPIN™, PYRAZINAMIDE™, ETHAMBUTOL™, RIFABUTIN™,CLARITHROMYCIN™, AZITHROMYCIN™, GANCICLOVIR™, FOSCARNET™, CIDOFOVIR™,FLUCONAZOLE™, ITRACONAZOLE™, KETOCONAZOLE™, ACYCLOVIR™, FAMCICOLVIR™,PYRIMETHAMINE™, LEUCOVORIN™, NEUPOGEN™ (filgrastim/G-CSF), and LEUKINE™(sargramostim/GM-CSF). In a specific embodiment, Therapeutics of theinvention are used in any combination withTRIMETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDINE™, and/orATOVAQUONE™ to prophylactically treat or prevent an opportunisticPneumocystis carinii pneumonia infection. In another specificembodiment, Therapeutics of the invention are used in any combinationwith ISONIAZID™, RIFAMPIN™, PYRAZINAMIDE™, and/or ETHAMBUTOL™ toprophylactically treat or prevent an opportunistic Mycobacterium aviumcomplex infection. In another specific embodiment, Therapeutics of theinvention are used in any combination with RIFABUTIN™, CLARITHROMYCIN™,and/or AZTHROMYCIN™ to prophylactically treat or prevent anopportunistic Mycobacterium tuberculosis infection. In another specificembodiment, Therapeutics of the invention are used in any combinationwith GANCICLOVIR™, FOSCARNET™, and/or CIDOFOVIR™ to prophylacticallytreat or prevent an opportunistic cytomegalovirus infection. In anotherspecific embodiment, Therapeutics of the invention are used in anycombination with FLUCONAZOLE™, ITRACONAZOLE™, and/or KETOCONAZOLE™ toprophylactically treat or prevent an opportunistic fungal infection. Inanother specific embodiment, Therapeutics of the invention are used inany combination with ACYCLOVIR™ and/or FAMCICOLVIR™ to prophylacticallytreat or prevent an opportunistic herpes simplex virus type I and/ortype II infection. In another specific embodiment, Therapeutics of theinvention are used in any combination with PYRIMETHAMINE™ and/orLEUCOVORIN™ to prophylactically treat or prevent an opportunisticToxoplasma gondii infection. In another specific embodiment,Therapeutics of the invention are used in any combination withLEUCOVORIN™ and/or NEUPOGEN™ to prophylactically treat or prevent anopportunistic bacterial infection.

[0547] In a further embodiment, the Therapeutics of the invention areadministered in combination with an antiviral agent. Antiviral agentsthat may be administered with the Therapeutics of the invention include,but are not limited to, acyclovir, ribavirin, amantadine, andremantidine.

[0548] In a further embodiment, the Therapeutics of the invention areadministered in combination with an antibiotic agent. Antibiotic agentsthat may be administered with the Therapeutics of the invention include,but are not limited to amoxicillin, beta- lactamases, aminoglycosides,beta-lactam (clycopeptide), beta-lactamases, Clindamycin,chloramphenicol, cephalosporins, ciprofloxacin, ciprofloxacin,erythromycin, fluoroquinolones, macrolides, metronidazole, penicillins,quinolones, rifampin, streptomycin, sulfonamide, tetracyclines,trimethoprim, trimethoprim- sulfamthoxazole, and vancomycin.

[0549] Conventional nonspecific immunosuppressive agents, that may beadministered in combination with the Therapeutics of the inventioninclude, but are not limited to steroids, cyclosporine, cyclosporineanalogs, cyclophosphamide methylprednisone, prednisone, azathioprine,FK-506, 15-deoxyspergualin, and other immunosuppressive agents that actby suppressing the function of responding T cells.

[0550] In specific embodiments, Therapeutics of the invention areadministered in combination with immunosuppressants. Immunosuppressantspreparations that may be administered with the Therapeutics of theinvention include, but are not limited to, ORTHOCLONE™ (OKT3),SANDIMMUNE™/NEORAL™/SANGDYA™ (cyclosporin), PROGRAF™ (tacrolimus),CELLCEPT™ (mycophenolate), Azathioprine, glucorticosteroids, andRAPAMUNE™ (sirolimus). In a specific embodiment, immunosuppressants maybe used to prevent rejection of organ or bone marrow transplantation.

[0551] In an additional embodiment, Therapeutics of the invention areadministered alone or in combination with one or more intravenous immuneglobulin preparations. Intravenous immune globulin preparations that maybe administered with the Therapeutics of the invention include, but notlimited to, GAMMAR™, IVEEGAM™, SANDOGLOBULIN™, GAMMAGARD S/D™, andGAMIMUNE™. In a specific embodiment, Therapeutics of the invention areadministered in combination with intravenous immune globulinpreparations in transplantation therapy (e.g., bone marrow transplant).

[0552] In an additional embodiment, the Therapeutics of the inventionare administered alone or in combination with an anti-inflammatoryagent. Anti-inflammatory agents that may be administered with theTherapeutics of the invention include, but are not limited to,glucocorticoids and the nonsteroidal anti-inflammatories,aminoarylcarboxylic acid derivatives, arylacetic acid derivatives,arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic acidderivatives, pyrazoles, pyrazolones, salicylic acid derivatives.thiazinecarboxamides, e-acetamidocaproic acid, S-adenosylmethionine,3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine,bucolome, difenpiramide, ditazol, emorfazone, guaiazulene, nabumetone,nimesulide, orgotein, oxaceprol, paranyline, perisoxal, pifoxime,proquazone, proxazole, and tenidap.

[0553] In another embodiment, compostions of the invention areadministered in combination with a chemotherapeutic agent.Chemotherapeutic agents that may be administered with the Therapeuticsof the invention include, but are not limited to, antibiotic derivatives(e.g., doxorubicin, bleomycin, daunorubicin, and dactinomycin);antiestrogens (e.g., tamoxifen); antimetabolites (e.g., fluorouracil,5-FU, methotrexate, floxuridine, interferon alpha-2b, glutamic acid,plicamycin, mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g.,carmustine, BCNU, lomustine, CCNU, cytosine arabinoside,cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin,busulfan, cis-platin, and vincristine sulfate); hormones (e.g.,medroxyprogesterone, estramustine phosphate sodium, ethinyl estradiol,estradiol, megestrol acetate, methyltestosterone, diethylstilbestroldiphosphate, chlorotrianisene, and testolactone); nitrogen mustardderivatives (e.g., mephalen, chorambucil, mechlorethamine (nitrogenmustard) and thiotepa); steroids and combinations (e.g., bethamethasonesodium phosphate); and others (e.g., dicarbazine, asparaginase,mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).

[0554] In a specific embodiment, Therapeutics of the invention areadministered in combination with CHOP (cyclophosphamide, doxorubicin,vincristine, and prednisone) or any combination of the components ofCHOP. In another embodiment, Therapeutics of the invention areadministered in combination with Rituximab. In a further embodiment,Therapeutics of the invention are administered with Rituxmab and CHOP,or Rituxmab and any combination of the components of CHOP.

[0555] In an additional embodiment, the Therapeutics of the inventionare administered in combination with cytokines. Cytokines that may beadministered with the Therapeutics of the invention include, but are notlimited to, IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13, IL15,anti-CD40, CD40L, IFN-gamma and TNF-alpha. In another embodiment,Therapeutics of the invention may be administered with any interleukin,including, but not limited to, IL-1alpha, IL-1beta, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15,IL-16, IL-17, IL-18, IL-19, IL-20, and IL-21.

[0556] In an additional embodiment, the Therapeutics of the inventionare administered in combination with angiogenic proteins. Angiogenicproteins that may be administered with the Therapeutics of the inventioninclude, but are not limited to, Glioma Derived Growth Factor (GDGF), asdisclosed in European Patent Number EP-399816; Platelet Derived GrowthFactor-A (PDGF-A), as disclosed in European Patent Number EP-6821 10;Platelet Derived Growth Factor-B (PDGF-B), as disclosed in EuropeanPatent Number EP-282317; Placental Growth Factor (PlGF), as disclosed inInternational Publication Number WO 92/06194; Placental Growth Factor-2(PIGF-2), as disclosed in Hauser et al., Gorwth Factors, 4:259-268(1993); Vascular Endothelial Growth Factor (VEGF), as disclosed inInternational Publication Number WO 90/13649; Vascular EndothelialGrowth Factor-A (VEGF-A), as disclosed in European Patent NumberEP-506477; Vascular Endothelial Growth Factor-2 (VEGF-2), as disclosedin International Publication Number WO 96/39515; Vascular EndothelialGrowth Factor B (VEGF-3); Vascular Endothelial Growth Factor B-186(VEGF- B186), as disclosed in International Publication Number WO96/26736; Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed inInternational Publication Number WO 98/02543; Vascular EndothelialGrowth Factor-D (VEGF-D), as disclosed in International PublicationNumber WO 98/07832; and Vascular Endothelial Growth Factor-E (VEGF-E),as disclosed in German Patent Number DE19639601. The above mentionedreferences are incorporated herein by reference herein.

[0557] In an additional embodiment, the Therapeutics of the inventionare administered in combination with hematopoietic growth factors.Hematopoietic growth factors that may be administered with theTherapeutics of the invention include, but are not limited to, LEUKINE™(SARGRAMOSTIM™) and NEUPOGEN™ (FILGRASTLM™).

[0558] In an additional embodiment, the Therapeutics of the inventionare administered in combination with Fibroblast Growth Factors.Fibroblast Growth Factors that may be administered with the Therapeuticsof the invention include, but are not limited to, FGF-1, FGF-2, FGF-3,FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12,FGF-13, FGF-14, and FGF-15.

[0559] In additional embodiments, the Therapeutics of the invention areadministered in combination with other therapeutic or prophylacticregimens, such as, for example, radiation therapy.

Example 12 Method of Treating Decreased Levels of the Polypeptide

[0560] The present invention relates to a method for treating anindividual in need of an increased level of a polypeptide of theinvention in the body comprising administering to such an individual acomposition comprising a therapeutically effective amount of an agonistof the invention (including polypeptides of the invention). Moreover, itwill be appreciated that conditions caused by a decrease in the standardor normal expression level of a secreted protein in an individual can betreated by administering the polypeptide of the present invention,preferably in the secreted form. Thus, the invention also provides amethod of treatment of an individual in need of an increased level ofthe polypeptide comprising administering to such an individual aTherapeutic comprising an amount of the polypeptide to increase theactivity level of the polypeptide in such an individual.

[0561] For example, a patient with decreased levels of a polypeptidereceives a daily dose 0.1-100 ug/kg of the polypeptide for sixconsecutive days. Preferably, the polypeptide is in the secreted form.The exact details of the dosing scheme, based on administration andformulation, are provided in the Examples.

Example 13 Method of Treating Increased Levels of the Polypeptide

[0562] The present invention also relates to a method of treating anindividual in need of a decreased level of a polypeptide of theinvention in the body comprising administering to such an individual acomposition comprising a therapeutically effective amount of anantagonist of the invention (including polypeptides and antibodies ofthe invention).

[0563] In one example, antisense technology is used to inhibitproduction of a polypeptide of the present invention. This technology isone example of a method of decreasing levels of a polypeptide,preferably a secreted form, due to a variety of etiologies, such ascancer. For example, a patient diagnosed with abnormally increasedlevels of a polypeptide is administered intravenously antisensepolynucleotides at 0.5, 1.0, 1.5, 2.0 and 3.0 mg/kg day for 21 days.This treatment is repeated after a 7-day rest period if the treatmentwas well tolerated. The formulation of the antisense polynucleotide isprovided in the Examples.

Example 14 Method of Treatment Using Gene Therapy-Ex Vivo

[0564] One method of gene therapy transplants fibroblasts, which arecapable of expressing a polypeptide, onto a patient. Generally,fibroblasts are obtained from a subject by skin biopsy. The resultingtissue is placed in tissue-culture medium and separated into smallpieces. Small chunks of the tissue are placed on a wet surface of atissue culture flask, approximately ten pieces are placed in each flask.The flask is turned upside down, closed tight and left at roomtemperature over night. After 24 hours at room temperature, the flask isinverted and the chunks of tissue remain fixed to the bottom of theflask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillinand streptomycin) is added. The flasks are then incubated at 37 degree Cfor approximately one week.

[0565] At this time, fresh media is added and subsequently changed everyseveral days. After an additional two weeks in culture, a monolayer offibroblasts emerge. The monolayer is trypsinized and scaled into largerflasks. pMV-7 (Kirschmeier, P. T. et al., DNA, 7:219-25 (1988)), flankedby the long terminal repeats of the Moloney murine sarcoma virus, isdigested with EcoRI and HindIll and subsequently treated with calfintestinal phosphatase. The linear vector is fractionated on agarose geland purified, using glass beads.

[0566] The cDNA encoding a polypeptide of the present invention can beamplified using PCR primers which correspond to the 5′ and 3′ endsequences respectively as set forth in Example 1 using primers andhaving appropriate restriction sites and initiation/stop codons, ifnecessary. Preferably, the 5′ primer contains an EcoRI site and the 3′primer includes a HindIII site. Equal quantities of the Moloney murinesarcoma virus linear backbone and the amplified EcoRI and HindIIIfragment are added together, in the presence of T4 DNA ligase. Theresulting mixture is maintained under conditions appropriate forligation of the two fragments. The ligation mixture is then used totransform bacteria HB101, which are then plated onto agar containingkanamycin for the purpose of confirming that the vector has the gene ofinterest properly inserted.

[0567] The amphotropic pA317 or GP+aml2 packaging cells are grown intissue culture to confluent density in Dulbecco's Modified Eagles Medium(DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSVvector containing the gene is then added to the media and the packagingcells transduced with the vector. The packaging cells now produceinfectious viral particles containing the gene (the packaging cells arenow referred to as producer cells).

[0568] Fresh media is added to the transduced producer cells, andsubsequently, the media is harvested from a 10 cm plate of confluentproducer cells. The spent media, containing the infectious viralparticles, is filtered through a millipore filter to remove detachedproducer cells and this media is then used to infect fibroblast cells.Media is removed from a sub-confluent plate of fibroblasts and quicklyreplaced with the media from the producer cells. This media is removedand replaced with fresh media. If the titer of virus is high, thenvirtually all fibroblasts will be infected and no selection is required.If the titer is very low, then it is necessary to use a retroviralvector that has a selectable marker, such as neo or his. Once thefibroblasts have been efficiently infected, the fibroblasts are analyzedto determine whether protein is produced.

[0569] The engineered fibroblasts are then transplanted onto the host,either alone or after having been grown to confluence on cytodex 3microcarrier beads.

Example 15 Gene Therapy Using Endogenous Genes Corresponding

[0570] To Polynucleotides of the Invention

[0571] Another method of gene therapy according to the present inventioninvolves operably associating the endogenous polynucleotide sequence ofthe invention with a promoter via homologous recombination as described,for example, in U.S. Pat. No. 5,641,670, issued Jun. 24, 1997;International Publication NO: WO 96/29411, published Sep. 26, 1996;International Publication NO: WO 94/12650, published Aug. 4, 1994;Koller et al., Proc. Natl. Acad. Sci. USA, 86:8932-8935 (1989); andZijlstra et al., Nature, 342:435-438 (1989). This method involves theactivation of a gene which is present in the target cells, but which isnot expressed in the cells, or is expressed at a lower level thandesired.

[0572] Polynucleotide constructs are made which contain a promoter andtargeting sequences, which are homologous to the 5′ non-coding sequenceof endogenous polynucleotide sequence, flanking the promoter. Thetargeting sequence will be sufficiently near the 5′ end of thepolynucleotide sequence so the promoter will be operably linked to theendogenous sequence upon homologous recombination. The promoter and thetargeting sequences can be amplified using PCR. Preferably, theamplified promoter contains distinct restriction enzyme sites on the 5′and 3′ ends. Preferably, the 3′ end of the first targeting sequencecontains the same restriction enzyme site as the 5′ end of the amplifiedpromoter and the 5′ end of the second targeting sequence contains thesame restriction site as the 3′ end of the amplified promoter.

[0573] The amplified promoter and the amplified targeting sequences aredigested with the appropriate restriction enzymes and subsequentlytreated with calf intestinal phosphatase. The digested promoter anddigested targeting sequences are added together in the presence of T4DNA ligase. The resulting mixture is maintained under conditionsappropriate for ligation of the two fragments. The construct is sizefractionated on an agarose gel then purified by phenol extraction andethanol precipitation.

[0574] In this Example, the polynucleotide constructs are administeredas naked polynucleotides via electroporation. However, thepolynucleotide constructs may also be administered withtransfection-facilitating agents, such as liposomes, viral sequences,viral particles, precipitating agents, etc. Such methods of delivery areknown in the art.

[0575] Once the cells are transfected, homologous recombination willtake place which results in the promoter being operably linked to theendogenous polynucleotide sequence. This results in the expression ofpolynucleotide corresponding to the polynucleotide in the cell.Expression may be detected by immunological staining, or any othermethod known in the art.

[0576] Fibroblasts are obtained from a subject by skin biopsy. Theresulting tissue is placed in DMEM+10% fetal calf serum. Exponentiallygrowing or early stationary phase fibroblasts are trypsinized and rinsedfrom the plastic surface with nutrient medium. An aliquot of the cellsuspension is removed for counting, and the remaining cells aresubjected to centrifugation. The supernatant is aspirated and the pelletis resuspended in 5 ml of electroporation buffer (20 mM HEPES pH 7.3,137 mM NaCl, 5 mM KCl, 0.7 mM Na₂ HPO₄, 6 mM dextrose). The cells arerecentrifuged, the supernatant aspirated, and the cells resuspended inelectroporation buffer containing 1 mg/ml acetylated bovine serumalbumin. The final cell suspension contains approximately 3×10⁶cells/ml. Electroporation should be performed immediately followingresuspension.

[0577] Plasmid DNA is prepared according to standard techniques. Forexample, to construct a plasmid for targeting to the locus correspondingto the polynucleotide of the invention, plasmid pUC18 (MBI Fermentas,Amherst, N.Y.) is digested with HindIII. The CMV promoter is amplifiedby PCR with an XbaI site on the 5′ end and a BamHI site on the 3′end.Two non-coding sequences are amplified via PCR: one non-coding sequence(fragment 1) is amplified with a HindIII site at the 5′ end and an Xbasite at the 3′end; the other non-coding sequence (fragment 2) isamplified with a BamHI site at the 5′end and a HindIII site at the3′end. The CMV promoter and the fragments (1 and 2) are digested withthe appropriate enzymes (CMV promoter—XbaI and BamHI; fragment 1—XbaI;fragment 2—BamHI) and ligated together. The resulting ligation productis digested with HindIII, and ligated with the HindIII-digested pUC18plasmid.

[0578] Plasmid DNA is added to a sterile cuvette with a 0.4 cm electrodegap (Bio-Rad). The final DNA concentration is generally at least 120μg/ml. 0.5 ml of the cell suspension (containing approximately 1.5×10⁶cells) is then added to the cuvette, and the cell suspension and DNAsolutions are gently mixed. Electroporation is performed with aGene-Pulser apparatus (Bio-Rad). Capacitance and voltage are set at 960μF and 250-300 V, respectively. As voltage increases, cell survivaldecreases, but the percentage of surviving cells that stably incorporatethe introduced DNA into their genome increases dramatically. Given theseparameters, a pulse time of approximately 14-20 mSec should be observed.

[0579] Electroporated cells are maintained at room temperature forapproximately 5 min, and the contents of the cuvette are then gentlyremoved with a sterile transfer pipette. The cells are added directly to10 ml of prewarmed nutrient media (DMEM with 15% calf serum) in a 10 cmdish and incubated at 37 degree C. The following day, the media isaspirated and replaced with 10 ml of fresh media and incubated for afurther 16-24 hours.

[0580] The engineered fibroblasts are then injected into the host,either alone or after having been grown to confluence on cytodex 3microcarrier beads. The fibroblasts now produce the protein product. Thefibroblasts can then be introduced into a patient as described above.

Example 16 Method of Treatment Using Gene Therapy—In Vivo

[0581] Another aspect of the present invention is using in vivo genetherapy methods to treat disorders, diseases and conditions. The genetherapy method relates to the introduction of naked nucleic acid (DNA,RNA, and antisense DNA or RNA) sequences into an animal to increase ordecrease the expression of the polypeptide. The polynucleotide of thepresent invention may be operatively linked to a promoter or any othergenetic elements necessary for the expression of the polypeptide by thetarget tissue. Such gene therapy and delivery techniques and methods areknown in the art, see, for example, WO90/11092, WO98/11779; U.S. Pat.Nos. 5,693,622, 5,705,151, 5,580,859; Tabata et al., Cardiovasc. Res.35(3):470-479 (1997); Chao et al., Pharmacol. Res. 35(6):517-522 (1997);Wolff, Neuromuscul. Disord. 7(5):314-318 (1997); Schwartz et al., GeneTher. 3(5):405-411 (1996); Tsurumi et al., Circulation 94(12):3281-3290(1996) (incorporated herein by reference).

[0582] The polynucleotide constructs may be delivered by any method thatdelivers injectable materials to the cells of an animal, such as,injection into the interstitial space of tissues (heart, muscle, skin,lung, liver, intestine and the like). The polynucleotide constructs canbe delivered in a pharmaceutically acceptable liquid or aqueous carrier.

[0583] The term “naked” polynucleotide, DNA or RNA, refers to sequencesthat are free from any delivery vehicle that acts to assist, promote, orfacilitate entry into the cell, including viral sequences, viralparticles, liposome formulations, lipofectin or to precipitating agentsand the like. However, the polynucleotides of the present invention mayalso be delivered in liposome formulations (such as those taught inFelgner P. L. et al. (1995) Ann. NY Acad. Sci. 772:126-139 and AbdallahB. et al. (1995) Biol. Cell 85(1):1-7) which can be prepared by methodswell known to those skilled in the art.

[0584] The polynucleotide vector constructs used in the gene therapymethod are preferably constructs that will not integrate into the hostgenome nor will they contain sequences that allow for replication. Anystrong promoter known to those skilled in the art can be used fordriving the expression of DNA. Unlike other gene therapies techniques,one major advantage of introducing naked nucleic acid sequences intotarget cells is the transitory nature of the polynucleotide synthesis inthe cells. Studies have shown that non-replicating DNA sequences can beintroduced into cells to provide production of the desired polypeptidefor periods of up to six months.

[0585] The polynucleotide construct can be delivered to the interstitialspace of tissues within the an animal, including of muscle, skin, brain,lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone,cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis,ovary, uterus, rectum, nervous system, eye, gland, and connectivetissue. Interstitial space of the tissues comprises the intercellularfluid, mucopolysaccharide matrix among the reticular fibers of organtissues, elastic fibers in the walls of vessels or chambers, collagenfibers of fibrous tissues, or that same matrix within connective tissueensheathing muscle cells or in the lacunae of bone. It is similarly thespace occupied by the plasma of the circulation and the lymph fluid ofthe lymphatic channels. Delivery to the interstitial space of muscletissue is preferred for the reasons discussed below. They may beconveniently delivered by injection into the tissues comprising thesecells. They are preferably delivered to and expressed in persistent,non-dividing cells which are differentiated, although delivery andexpression may be achieved in non-differentiated or less completelydifferentiated cells, such as, for example, stem cells of blood or skinfibroblasts. In vivo muscle cells are particularly competent in theirability to take up and express polynucleotides.

[0586] For the naked polynucleotide injection, an effective dosageamount of DNA or RNA will be in the range of from about 0.05 g/kg bodyweight to about 50 mg/kg body weight. Preferably the dosage will be fromabout 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05mg/kg to about 5 mg/kg. Of course, as the artisan of ordinary skill willappreciate, this dosage will vary according to the tissue site ofinjection. The appropriate and effective dosage of nucleic acid sequencecan readily be determined by those of ordinary skill in the art and maydepend on the condition being treated and the route of administration.The preferred route of administration is by the parenteral route ofinjection into the interstitial space of tissues. However, otherparenteral routes may also be used, such as, inhalation of an aerosolformulation particularly for delivery to lungs or bronchial tissues,throat or mucous membranes of the nose. In addition, nakedpolynucleotide constructs can be delivered to arteries duringangioplasty by the catheter used in the procedure.

[0587] The dose response effects of injected polynucleotide in muscle invivo is determined as follows. Suitable template DNA for production ofmRNA coding for polypeptide of the present invention is prepared inaccordance with a standard recombinant DNA methodology. The templateDNA, which may be either circular or linear, is either used as naked DNAor complexed with liposomes. The quadriceps muscles of mice are theninjected with various amounts of the template DNA.

[0588] Five to six week old female and male Balb/C mice are anesthetizedby intraperitoneal injection with 0.3 ml of 2.5% Avertin. A 1.5 cmincision is made on the anterior thigh, and the quadriceps muscle isdirectly visualized. The template DNA is injected in 0.1 ml of carrierin a 1 cc syringe through a 27 gauge needle over one minute,approximately 0.5 cm from the distal insertion site of the muscle intothe knee and about 0.2 cm deep. A suture is placed over the injectionsite for future localization, and the skin is closed with stainlesssteel clips.

[0589] After an appropriate incubation time (e.g., 7 days) muscleextracts are prepared by excising the entire quadriceps. Every fifth 15um cross-section of the individual quadriceps muscles is histochemicallystained for protein expression. A time course for protein expression maybe done in a similar fashion except that quadriceps from different miceare harvested at different times. Persistence of DNA in muscle followinginjection may be determined by Southern blot analysis after preparingtotal cellular DNA and HIRT supernatants from injected and control mice.The results of the above experimentation in mice can be use toextrapolate proper dosages and other treatment parameters in humans andother animals using naked DNA.

Example 17 Transgenic Animals.

[0590] The polypeptides of the invention can also be expressed intransgenic animals. Animals of any species, including, but not limitedto, mice, rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats,sheep, cows and non-human primates, e.g., baboons, monkeys, andchimpanzees may be used to generate transgenic animals. In a specificembodiment, techniques described herein or otherwise known in the art,are used to express polypeptides of the invention in humans, as part ofa gene therapy protocol.

[0591] Any technique known in the art may be used to introduce thetransgene (i.e., polynucleotides of the invention) into animals toproduce the founder lines of transgenic animals. Such techniquesinclude, but are not limited to, pronuclear microinjection (Paterson etal., Appl. Microbiol. Biotechnol. 40:691-698 (1994); Carver et al.,Biotechnology (NY) 11:1263-1270 (1993); Wright et al., Biotechnology(NY) 9:830-834 (1991); and Hoppe et al., U.S. Pat. No. 4,873,191(1989)); retrovirus mediated gene transfer into germ lines (Van derPutten et al., Proc. Natl. Acad. Sci., USA 82:6148-6152 (1985)),blastocysts or embryos; gene targeting in embryonic stem cells (Thompsonet al., Cell 56:313-321 (1989)); electroporation of cells or embryos(Lo, 1983, Mol Cell. Biol. 3:1803-1814 (1983)); introduction of thepolynucleotides of the invention using a gene gun (see, e.g., Ulmer etal., Science 259:1745 (1993); introducing nucleic acid constructs intoembryonic pleuripotent stem cells and transferring the stem cells backinto the blastocyst; and sperm-mediated gene transfer (Lavitrano et al.,Cell 57:717-723 (1989); etc. For a review of such techniques, seeGordon, “Transgenic Animals,” Intl. Rev. Cytol. 115:171-229 (1989),which is incorporated by reference herein in its entirety.

[0592] Any technique known in the art may be used to produce transgenicclones containing polynucleotides of the invention, for example, nucleartransfer into enucleated oocytes of nuclei from cultured embryonic,fetal, or adult cells induced to quiescence (Campell et al., Nature380:64-66 (1996); Wilmut et al., Nature 385:810-813 (1997)).

[0593] The present invention provides for transgenic animals that carrythe transgene in all their cells, as well as animals which carry thetransgene in some, but not all their cells, i.e., mosaic animals orchimeric. The transgene may be integrated as a single transgene or asmultiple copies such as in concatamers, e.g., head-to-head tandems orhead-to-tail tandems. The transgene may also be selectively introducedinto and activated in a particular cell type by following, for example,the teaching of Lasko et al. (Lasko et al., Proc. Natl. Acad. Sci. USA89:6232-6236 (1992)). The regulatory sequences required for such acell-type specific activation will depend upon the particular cell typeof interest, and will be apparent to those of skill in the art. When itis desired that the polynucleotide transgene be integrated into thechromosomal site of the endogenous gene, gene targeting is preferred.Briefly, when such a technique is to be utilized, vectors containingsome nucleotide sequences homologous to the endogenous gene are designedfor the purpose of integrating, via homologous recombination withchromosomal sequences, into and disrupting the function of thenucleotide sequence of the endogenous gene. The transgene may also beselectively introduced into a particular cell type, thus inactivatingthe endogenous gene in only that cell type, by following, for example,the teaching of Gu et al. (Gu et al., Science 265:103-106 (1994)). Theregulatory sequences required for such a cell-type specific inactivationwill depend upon the particular cell type of interest, and will beapparent to those of skill in the art.

[0594] Once transgenic animals have been generated, the expression ofthe recombinant gene may be assayed utilizing standard techniques.Initial screening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to verify that integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques which include, but are not limited to, Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and reverse transcriptase-PCR (rt-PCR). Samples of transgenicgene-expressing tissue may also be evaluated immunocytochemically orimmunohistochemically using antibodies specific for the transgeneproduct.

[0595] Once the founder animals are produced, they may be bred, inbred,outbred, or crossbred to produce colonies of the particular animal.Examples of such breeding strategies include, but are not limited to:outbreeding of founder animals with more than one integration site inorder to establish separate lines; inbreeding of separate lines in orderto produce compound transgenics that express the transgene at higherlevels because of the effects of additive expression of each transgene;crossing of heterozygous transgenic animals to produce animalshomozygous for a given integration site in order to both augmentexpression and eliminate the need for screening of animals by DNAanalysis; crossing of separate homozygous lines to produce compoundheterozygous or homozygous lines; and breeding to place the transgene ona distinct background that is appropriate for an experimental model ofinterest.

[0596] Transgenic animals of the invention have uses which include, butare not limited to, animal model systems useful in elaborating thebiological function of polypeptides of the present invention, studyingdiseases, disorders, and/or conditions associated with aberrantexpression, and in screening for compounds effective in amelioratingsuch diseases, disorders, and/or conditions.

Example 18 Knock-Out Animals

[0597] Endogenous gene expression can also be reduced by inactivating or“knocking out” the gene and/or its promoter using targeted homologousrecombination. (E.g., see Smithies et al., Nature 317:230-234 (1985);Thomas & Capecchi, Cell 51:503-512 (1987); Thompson et al., Cell5:313-321 (1989); each of which is incorporated by reference herein inits entirety). For example, a mutant, non-functional polynucleotide ofthe invention (or a completely unrelated DNA sequence) flanked by DNAhomologous to the endogenous polynucleotide sequence (either the codingregions or regulatory regions of the gene) can be used, with or withouta selectable marker and/or a negative selectable marker, to transfectcells that express polypeptides of the invention in vivo. In anotherembodiment, techniques known in the art are used to generate knockoutsin cells that contain, but do not express the gene of interest.Insertion of the DNA construct, via targeted homologous recombination,results in inactivation of the targeted gene. Such approaches areparticularly suited in research and agricultural fields wheremodifications to embryonic stem cells can be used to generate animaloffspring with an inactive targeted gene (e.g., see Thomas & Capecchi1987 and Thompson 1989, supra). However this approach can be routinelyadapted for use in humans provided the recombinant DNA constructs aredirectly administered or targeted to the required site in vivo usingappropriate viral vectors that will be apparent to those of skill in theart.

[0598] In further embodiments of the invention, cells that aregenetically engineered to express the polypeptides of the invention, oralternatively, that are genetically engineered not to express thepolypeptides of the invention (e.g., knockouts) are administered to apatient in vivo. Such cells may be obtained from the patient (i.e.,animal, including human) or an MHC compatible donor and can include, butare not limited to fibroblasts, bone marrow cells, blood cells (e.g.,lymphocytes), adipocytes, muscle cells, endothelial cells etc. The cellsare genetically engineered in vitro using recombinant DNA techniques tointroduce the coding sequence of polypeptides of the invention into thecells, or alternatively, to disrupt the coding sequence and/orendogenous regulatory sequence associated with the polypeptides of theinvention, e.g., by transduction (using viral vectors, and preferablyvectors that integrate the transgene into the cell genome) ortransfection procedures, including, but not limited to, the use ofplasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. Thecoding sequence of the polypeptides of the invention can be placed underthe control of a strong constitutive or inducible promoter orpromoter/enhancer to achieve expression, and preferably secretion, ofthe polypeptides of the invention. The engineered cells which expressand preferably secrete the polypeptides of the invention can beintroduced into the patient systemically, e.g., in the circulation, orintraperitoneally.

[0599] Alternatively, the cells can be incorporated into a matrix andimplanted in the body, e.g., genetically engineered fibroblasts can beimplanted as part of a skin graft; genetically engineered endothelialcells can be implanted as part of a lymphatic or vascular graft. (See,for example, Anderson et al. U.S. Pat. No. 5,399,349; and Mulligan &Wilson, U.S. Pat. No. 5,460,959 each of which is incorporated byreference herein in its entirety).

[0600] When the cells to be administered are non-autologous or non-MHCcompatible cells, they can be administered using well known techniqueswhich prevent the development of a host immune response against theintroduced cells. For example, the cells may be introduced in anencapsulated form which, while allowing for an exchange of componentswith the immediate extracellular environment, does not allow theintroduced cells to be recognized by the host immune system.

[0601] Transgenic and “knock-out” animals of the invention have useswhich include, but are not limited to, animal model systems useful inelaborating the biological function of polypeptides of the presentinvention, studying diseases, disorders, and/or conditions associatedwith aberrant expression, and in screening for compounds effective inameliorating such diseases, disorders, and/or conditions.

Example 19 Biological Effects of Polypeptides of the Invention

[0602] Astrocyte and Neuronal Assays

[0603] Recombinant polypeptides of the invention, expressed inEscherichia coli and purified as described above, can be tested foractivity in promoting the survival, neurite outgrowth, or phenotypicdifferentiation of cortical neuronal cells and for inducing theproliferation of glial fibrillary acidic protein immunopositive cells,astrocytes. The selection of cortical cells for the bioassay is based onthe prevalent expression of FGF-1 and FGF-2 in cortical structures andon the previously reported enhancement of cortical neuronal survivalresulting from FCF-2 treatment. A thymidine incorporation assay, forexample, can be used to elucidate a polypeptide of the invention'sactivity on these cells. Moreover, previous reports describing thebiological effects of FGF-2 (basic FGF) on cortical or hippocampalneurons in vitro have demonstrated increases in both neuron survival andneurite outgrowth (Walicke et al., “Fibroblast growth factor promotessurvival of dissociated hippocampal neurons and enhances neuriteextension.” Proc. Natl. Acad. Sci. USA 83:3012-3016. (1986), assayherein incorporated by reference in its entirety). However, reports fromexperiments done on PC-12 cells suggest that these two responses are notnecessarily synonymous and may depend on not only which FGF is beingtested but also on which receptor(s) are expressed on the target cells.Using the primary cortical neuronal culture paradigm, the ability of apolypeptide of the invention to induce neurite outgrowth can be comparedto the response achieved with FGF-2 using, for example, a thymidineincorporation assay.

[0604] Fibroblast and Endothelial Cell Assays

[0605] Human lung fibroblasts are obtained from Clonetics (San Diego,Calif.) and maintained in growth media from Clonetics. Dermalmicrovascular endothelial cells are obtained from Cell Applications (SanDiego, Calif.). For proliferation assays, the human lung fibroblasts anddermal microvascular endothelial cells can be cultured at 5,000cells/well in a 96-well plate for one day in growth medium. The cellsare then incubated for one day in 0.1% BSA basal medium. After replacingthe medium with fresh 0.1% BSA medium, the cells are incubated with thetest proteins for 3 days. Alamar Blue (Alamar Biosciences, Sacramento,Calif.) is added to each well to a final concentration of 10%. The cellsare incubated for 4 hr. Cell viability is measured by reading in aCytoFluor fluorescence reader. For the PGE₂ assays, the human lungfibroblasts are cultured at 5,000 cells/well in a 96-well plate for oneday. After a medium change to 0.1% BSA basal medium, the cells areincubated with FGF-2 or polypeptides of the invention with or withoutIL-1α for 24 hours. The supernatants are collected and assayed for PGE₂by EIA kit (Cayman, Ann Arbor, MI). For the IL-6 assays, the human lungfibroblasts are cultured at 5,000 cells/well in a 96-well plate for oneday. After a medium change to 0.1% BSA basal medium, the cells areincubated with FGF-2 or with or without polypeptides of the inventionIL-1α for 24 hours. The supernatants are collected and assayed for IL-6by ELISA kit (Endogen, Cambridge, Mass.).

[0606] Human lung fibroblasts are cultured with FGF-2 or polypeptides ofthe invention for 3 days in basal medium before the addition of AlamarBlue to assess effects on growth of the fibroblasts. FGF-2 should show astimulation at 10-2500 ng/ml which can be used to compare stimulationwith polypeptides of the invention.

[0607] Parkinson Models.

[0608] The loss of motor function in Parkinson's disease is attributedto a deficiency of striatal dopamine resulting from the degeneration ofthe nigrostriatal dopaminergic projection neurons. An animal model forParkinson's that has been extensively characterized involves thesystemic administration of 1-methyl-4 phenyl 1,2,3,6-tetrahydropyridine(MPTP). In the CNS, MPTP is taken-up by astrocytes and catabolized bymonoamine oxidase B to 1-methyl-4-phenyl pyridine (MPP+) and released.Subsequently, MPP+is actively accumulated in dopaminergic neurons by thehigh-affinity reuptake transporter for dopamine. MPP⁺ is thenconcentrated in mitochondria by the electrochemical gradient andselectively inhibits nicotidamide adenine disphosphate: ubiquinoneoxidoreductionase (complex I), thereby interfering with electrontransport and eventually generating oxygen radicals.

[0609] It has been demonstrated in tissue culture paradigms that FGF-2(basic FGF) has trophic activity towards nigral dopaminergic neurons(Ferrari et al., Dev. Biol. 1989). Recently, Dr. Unsicker's group hasdemonstrated that administering FGF-2 in gel foam implants in thestriatum results in the near complete protection of nigral dopaminergicneurons from the toxicity associated with MPTP exposure (Otto andUnsicker, J. Neuroscience, 1990).

[0610] Based on the data with FGF-2, polypeptides of the invention canbe evaluated to determine whether it has an action similar to that ofFGF-2 in enhancing dopaminergic neuronal survival in vitro and it canalso be tested in vivo for protection of dopaminergic neurons in thestriatum from the damage associated with MPTP treatment. The potentialeffect of a polypeptide of the invention is first examined in vitro in adopaminergic neuronal cell culture paradigm. The cultures are preparedby dissecting the midbrain floor plate from gestation day 14 Wistar ratembryos. The tissue is dissociated with trypsin and seeded at a densityof 200,000 cells/cm² on polyorthinine-laminin coated glass coverslips.The cells are maintained in Dulbecco's Modified Eagle's medium and F12medium containing hormonal supplements (N1). The cultures are fixed withparaformaldehyde after 8 days in vitro and are processed for tyrosinehydroxylase, a specific marker for dopminergic neurons,immunohistochemical staining. Dissociated cell cultures are preparedfrom embryonic rats. The culture medium is changed every third day andthe factors are also added at that time.

[0611] Since the dopaminergic neurons are isolated from animals atgestation day 14, a developmental time which is past the stage when thedopaminergic precursor cells are proliferating, an increase in thenumber of tyrosine hydroxylase immunopositive neurons would represent anincrease in the number of dopaminergic neurons surviving in vitro.Therefore, if a polypeptide of the invention acts to prolong thesurvival of dopaminergic neurons, it would suggest that the polypeptidemay be involved in Parkinson's Disease.

[0612] The studies described in this example tested activity of apolypeptide of the invention. However, one skilled in the art couldeasily modify the exemplified studies to test the activity ofpolynucleotides (e.g., gene therapy), agonists, and/or antagonists ofthe invention.

[0613] The entire disclosure of each document cited (including patents,patent applications, journal articles, abstracts, laboratory manuals,books, or other disclosures) in the Background of the Invention,Detailed Description, and Examples is hereby incorporated herein byreference.

1 21 1 1564 DNA Homo sapiens CDS (89)..(1318) 1 gagcggagcg gagcacagtccgccgagcac aagctccagc atcccgtcag gggttgcagg 60 tgtgtgggag gcttgaaactgttacaat atg gct ttc ctt gga ctc ttc tct 112 Met Ala Phe Leu Gly Leu PheSer 1 5 ttg ctg gtt ctg caa agt atg gct aca ggg gcc act ttc cct gag gaa160 Leu Leu Val Leu Gln Ser Met Ala Thr Gly Ala Thr Phe Pro Glu Glu 1015 20 gcc att gct gac ttg tca gtg aat atg tat aat cgt ctt aga gcc act208 Ala Ile Ala Asp Leu Ser Val Asn Met Tyr Asn Arg Leu Arg Ala Thr 2530 35 40 ggt gaa gat gaa aat att ctc ttc tct cca ttg agt att gct ctt gca256 Gly Glu Asp Glu Asn Ile Leu Phe Ser Pro Leu Ser Ile Ala Leu Ala 4550 55 atg gga atg atg gaa ctt ggg gcc caa gga tct acc cag aaa gaa atc304 Met Gly Met Met Glu Leu Gly Ala Gln Gly Ser Thr Gln Lys Glu Ile 6065 70 cgc cac tca atg gga tat gac agc cta aaa aat ggt gaa gaa ttt tct352 Arg His Ser Met Gly Tyr Asp Ser Leu Lys Asn Gly Glu Glu Phe Ser 7580 85 ttc ttg aag gag ttt tca aac atg gta act gct aaa gag agc caa tat400 Phe Leu Lys Glu Phe Ser Asn Met Val Thr Ala Lys Glu Ser Gln Tyr 9095 100 gtg atg aaa att gcc aat tcc ttg ttt gtg caa aat gga ttt cat gtc448 Val Met Lys Ile Ala Asn Ser Leu Phe Val Gln Asn Gly Phe His Val 105110 115 120 aat gag gag ttt ttg caa atg atg aaa aaa tat ttt aat gca gcagta 496 Asn Glu Glu Phe Leu Gln Met Met Lys Lys Tyr Phe Asn Ala Ala Val125 130 135 aat cat gtg gac ttc agt caa aat gta gcc gtg gcc aac tac atcaat 544 Asn His Val Asp Phe Ser Gln Asn Val Ala Val Ala Asn Tyr Ile Asn140 145 150 aag tgg gtg gag aat aac aca aac aat ctg gtg aaa gat ttg gtatcc 592 Lys Trp Val Glu Asn Asn Thr Asn Asn Leu Val Lys Asp Leu Val Ser155 160 165 cca agg gat ttt gat gct gcc act tat ctg gcc ctc att aat gctgtc 640 Pro Arg Asp Phe Asp Ala Ala Thr Tyr Leu Ala Leu Ile Asn Ala Val170 175 180 tat ttc aag ggg aac tgg aag tcg cag ttt agg cct gaa aat actaga 688 Tyr Phe Lys Gly Asn Trp Lys Ser Gln Phe Arg Pro Glu Asn Thr Arg185 190 195 200 acc ttt tct ttc act aaa gat gat gaa agt gaa gtc caa attcca atg 736 Thr Phe Ser Phe Thr Lys Asp Asp Glu Ser Glu Val Gln Ile ProMet 205 210 215 atg tat cag caa gga gaa ttt tat tat ggg gaa ttt agt gatggc tcc 784 Met Tyr Gln Gln Gly Glu Phe Tyr Tyr Gly Glu Phe Ser Asp GlySer 220 225 230 aat gaa gct ggt ggt atc tac caa gtc cta gaa ata cca tatgaa gga 832 Asn Glu Ala Gly Gly Ile Tyr Gln Val Leu Glu Ile Pro Tyr GluGly 235 240 245 gat gaa ata agc atg atg ctg gtg ctg tcc aga cag gaa gttcct ctt 880 Asp Glu Ile Ser Met Met Leu Val Leu Ser Arg Gln Glu Val ProLeu 250 255 260 gct act ctg gag cca tta gtc aaa gca cag ctg gtt gaa gaatgg gca 928 Ala Thr Leu Glu Pro Leu Val Lys Ala Gln Leu Val Glu Glu TrpAla 265 270 275 280 aac tct gtg aag aag caa aaa gta gaa gta tac ctg cccagg ttc aca 976 Asn Ser Val Lys Lys Gln Lys Val Glu Val Tyr Leu Pro ArgPhe Thr 285 290 295 gtg gaa cag gaa att gat tta aaa gat gtt ttg aag gctctt gga ata 1024 Val Glu Gln Glu Ile Asp Leu Lys Asp Val Leu Lys Ala LeuGly Ile 300 305 310 act gaa att ttc atc aaa gat gca aat ttg aca ggc ctctct gat aat 1072 Thr Glu Ile Phe Ile Lys Asp Ala Asn Leu Thr Gly Leu SerAsp Asn 315 320 325 aag gag att ttt ctt tcc aaa gca att cac aag tcc ttccta gag gtt 1120 Lys Glu Ile Phe Leu Ser Lys Ala Ile His Lys Ser Phe LeuGlu Val 330 335 340 aat gaa gaa ggc tca gaa gct gct gct gtc tca gga atgatt gca att 1168 Asn Glu Glu Gly Ser Glu Ala Ala Ala Val Ser Gly Met IleAla Ile 345 350 355 360 agt agg atg gct gtg ctg tat cct caa gtt att gtcgac cat cca ttt 1216 Ser Arg Met Ala Val Leu Tyr Pro Gln Val Ile Val AspHis Pro Phe 365 370 375 ttc ttt ctt atc aga aac agg aga act ggt aca attcta ttc atg gga 1264 Phe Phe Leu Ile Arg Asn Arg Arg Thr Gly Thr Ile LeuPhe Met Gly 380 385 390 cga gtc atg cat cct gaa aca atg aac aca agt ggacat gat ttc gaa 1312 Arg Val Met His Pro Glu Thr Met Asn Thr Ser Gly HisAsp Phe Glu 395 400 405 gaa ctt taagttactt tatttgaata acaaggaaaacagtaactaa gcacattatg 1368 Glu Leu 410 tttgcaactg gtatatattt aggatttgtgttttacagta tatcttaaga taatatttaa 1428 aatagttcca gataaaaaca atatatgtaaattataagta acttgtcaag gaatgttatc 1488 agtattaagc taatggtcct gttatgtcattgtgtttgtg tgctgttgtt taaaataaaa 1548 gtacctattg aacatg 1564 2 410 PRTHomo sapiens 2 Met Ala Phe Leu Gly Leu Phe Ser Leu Leu Val Leu Gln SerMet Ala 1 5 10 15 Thr Gly Ala Thr Phe Pro Glu Glu Ala Ile Ala Asp LeuSer Val Asn 20 25 30 Met Tyr Asn Arg Leu Arg Ala Thr Gly Glu Asp Glu AsnIle Leu Phe 35 40 45 Ser Pro Leu Ser Ile Ala Leu Ala Met Gly Met Met GluLeu Gly Ala 50 55 60 Gln Gly Ser Thr Gln Lys Glu Ile Arg His Ser Met GlyTyr Asp Ser 65 70 75 80 Leu Lys Asn Gly Glu Glu Phe Ser Phe Leu Lys GluPhe Ser Asn Met 85 90 95 Val Thr Ala Lys Glu Ser Gln Tyr Val Met Lys IleAla Asn Ser Leu 100 105 110 Phe Val Gln Asn Gly Phe His Val Asn Glu GluPhe Leu Gln Met Met 115 120 125 Lys Lys Tyr Phe Asn Ala Ala Val Asn HisVal Asp Phe Ser Gln Asn 130 135 140 Val Ala Val Ala Asn Tyr Ile Asn LysTrp Val Glu Asn Asn Thr Asn 145 150 155 160 Asn Leu Val Lys Asp Leu ValSer Pro Arg Asp Phe Asp Ala Ala Thr 165 170 175 Tyr Leu Ala Leu Ile AsnAla Val Tyr Phe Lys Gly Asn Trp Lys Ser 180 185 190 Gln Phe Arg Pro GluAsn Thr Arg Thr Phe Ser Phe Thr Lys Asp Asp 195 200 205 Glu Ser Glu ValGln Ile Pro Met Met Tyr Gln Gln Gly Glu Phe Tyr 210 215 220 Tyr Gly GluPhe Ser Asp Gly Ser Asn Glu Ala Gly Gly Ile Tyr Gln 225 230 235 240 ValLeu Glu Ile Pro Tyr Glu Gly Asp Glu Ile Ser Met Met Leu Val 245 250 255Leu Ser Arg Gln Glu Val Pro Leu Ala Thr Leu Glu Pro Leu Val Lys 260 265270 Ala Gln Leu Val Glu Glu Trp Ala Asn Ser Val Lys Lys Gln Lys Val 275280 285 Glu Val Tyr Leu Pro Arg Phe Thr Val Glu Gln Glu Ile Asp Leu Lys290 295 300 Asp Val Leu Lys Ala Leu Gly Ile Thr Glu Ile Phe Ile Lys AspAla 305 310 315 320 Asn Leu Thr Gly Leu Ser Asp Asn Lys Glu Ile Phe LeuSer Lys Ala 325 330 335 Ile His Lys Ser Phe Leu Glu Val Asn Glu Glu GlySer Glu Ala Ala 340 345 350 Ala Val Ser Gly Met Ile Ala Ile Ser Arg MetAla Val Leu Tyr Pro 355 360 365 Gln Val Ile Val Asp His Pro Phe Phe PheLeu Ile Arg Asn Arg Arg 370 375 380 Thr Gly Thr Ile Leu Phe Met Gly ArgVal Met His Pro Glu Thr Met 385 390 395 400 Asn Thr Ser Gly His Asp PheGlu Glu Leu 405 410 3 410 PRT Gallus gallus 3 Met Tyr Phe Leu Gly LeuLeu Ser Leu Leu Val Leu Pro Ser Lys Ala 1 5 10 15 Phe Lys Thr Asn PhePro Asp Glu Thr Ile Ala Glu Leu Ser Val Asn 20 25 30 Val Tyr Asn Gln LeuArg Ala Ala Arg Glu Asp Glu Asn Ile Leu Phe 35 40 45 Cys Pro Leu Ser IleAla Ile Ala Met Gly Met Ile Glu Leu Gly Ala 50 55 60 His Gly Thr Thr LeuLys Glu Ile Arg His Ser Leu Gly Phe Asp Ser 65 70 75 80 Leu Lys Asn GlyGlu Glu Phe Thr Phe Leu Lys Asp Leu Ser Asp Met 85 90 95 Ala Thr Thr GluGlu Ser His Tyr Val Leu Asn Met Ala Asn Ser Leu 100 105 110 Tyr Val GlnAsn Gly Phe His Val Ser Glu Lys Phe Leu Gln Leu Val 115 120 125 Lys LysTyr Phe Lys Ala Glu Val Glu Asn Ile Asp Phe Ser Gln Ser 130 135 140 AlaAla Val Ala Thr His Ile Asn Lys Trp Val Glu Asn His Thr Asn 145 150 155160 Asn Met Ile Lys Asp Phe Val Ser Ser Arg Asp Phe Ser Ala Leu Thr 165170 175 His Leu Val Leu Ile Asn Ala Ile Tyr Phe Lys Gly Asn Trp Lys Ser180 185 190 Gln Phe Arg Pro Glu Asn Thr Arg Thr Phe Ser Phe Thr Lys AspAsp 195 200 205 Glu Thr Glu Val Gln Ile Pro Met Met Tyr Gln Gln Gly GluPhe Tyr 210 215 220 Tyr Gly Glu Phe Ser Asp Gly Ser Asn Glu Ala Gly GlyIle Tyr Gln 225 230 235 240 Val Leu Glu Ile Pro Tyr Glu Gly Asp Glu IleSer Met Met Ile Val 245 250 255 Leu Ser Arg Gln Glu Val Pro Leu Val ThrLeu Glu Pro Leu Val Lys 260 265 270 Ala Ser Leu Ile Asn Glu Trp Ala AsnSer Val Lys Lys Gln Lys Val 275 280 285 Glu Val Tyr Leu Pro Arg Phe ThrVal Glu Gln Glu Ile Asp Leu Lys 290 295 300 Asp Val Leu Lys Gly Leu GlyIle Thr Glu Val Phe Ser Arg Ser Ala 305 310 315 320 Asp Leu Thr Ala MetSer Asp Asn Lys Glu Leu Tyr Leu Ala Lys Ala 325 330 335 Phe His Lys AlaPhe Leu Glu Val Asn Glu Glu Gly Ser Glu Ala Ala 340 345 350 Ala Ala SerGly Met Ile Ala Ile Ser Arg Met Ala Val Leu Tyr Pro 355 360 365 Gln ValIle Val Asp His Pro Phe Phe Phe Leu Val Arg Asn Arg Arg 370 375 380 ThrGly Thr Val Leu Phe Met Gly Arg Val Met His Pro Glu Ala Met 385 390 395400 Asn Thr Ser Gly His Asp Phe Glu Glu Leu 405 410 4 402 PRT Bos taurus4 Met Arg Met Ser Pro Val Phe Ala Cys Leu Ala Leu Gly Leu Ala Leu 1 5 1015 Ile Phe Gly Glu Gly Ser Ala Ser Tyr Gln Pro Gln Ser Ala Ala Ala 20 2530 Ser Leu Ala Thr Asp Phe Gly Val Lys Val Phe Gln Gln Val Val Arg 35 4045 Ala Ser Lys Asp Arg Asn Val Val Phe Ser Pro Tyr Gly Val Ala Ser 50 5560 Val Leu Ala Met Leu Gln Leu Thr Thr Gly Gly Glu Thr Arg Gln Gln 65 7075 80 Ile Gln Glu Ala Met Gln Phe Lys Ile Glu Glu Lys Gly Met Ala Pro 8590 95 Ala Phe His Arg Leu Tyr Lys Glu Leu Met Gly Pro Trp Asn Lys Asp100 105 110 Glu Ile Ser Thr Ala Asp Ala Ile Phe Val Gln Arg Asp Leu GluLeu 115 120 125 Val His Gly Phe Met Pro Asn Phe Phe Arg Leu Phe Arg ThrThr Val 130 135 140 Lys Gln Val Asp Phe Ser Glu Val Glu Arg Ala Arg PheIle Val Asn 145 150 155 160 Asp Trp Val Lys Arg His Thr Lys Gly Met IleSer Asp Leu Leu Gly 165 170 175 Glu Gly Ala Val Asp Gln Leu Thr Arg LeuVal Leu Val Asn Ala Leu 180 185 190 Tyr Phe Asn Gly Gln Trp Lys Met ProPhe Pro Glu Ser Asn Thr His 195 200 205 His Arg Leu Phe His Lys Ser AspGly Ser Thr Ile Ser Val Pro Met 210 215 220 Met Ala Gln Thr Asn Lys PheAsn Tyr Thr Glu Phe Thr Thr Pro Asp 225 230 235 240 Gly Arg Tyr Tyr AspIle Leu Glu Leu Pro Tyr His Gly Asn Thr Leu 245 250 255 Ser Met Leu IleAla Ala Pro Tyr Glu Lys Glu Val Pro Leu Ser Ala 260 265 270 Leu Thr SerIle Leu Asp Ala Glu Leu Ile Ser Gln Trp Lys Gly Asn 275 280 285 Met ThrArg Leu Thr Arg Leu Leu Val Leu Pro Lys Phe Ser Leu Glu 290 295 300 ThrGlu Ile Asp Leu Arg Arg Pro Leu Glu Asn Leu Gly Met Thr Asp 305 310 315320 Met Phe Arg Pro Ser Gln Ala Asp Phe Ser Ser Phe Ser Asp Gln Glu 325330 335 Phe Leu Tyr Val Ser Gln Ala Leu Gln Lys Val Lys Ile Glu Val Asn340 345 350 Glu Ser Gly Thr Leu Ala Ser Ser Ser Thr Ala Leu Val Val SerAla 355 360 365 Arg Met Ala Pro Glu Glu Ile Ile Met Asp Arg Pro Phe LeuPhe Val 370 375 380 Val Arg His Asn Pro Thr Gly Thr Val Leu Phe Met GlyGln Val Met 385 390 395 400 Glu Pro 5 397 PRT Rattus norvegicus 5 MetAsn Trp His Phe Pro Phe Phe Ile Leu Thr Thr Val Thr Leu Ser 1 5 10 15Ser Val Tyr Ser Gln Leu Asn Ser Leu Ser Leu Glu Glu Leu Gly Ser 20 25 30Asp Thr Gly Ile Gln Val Phe Asn Gln Ile Ile Lys Ser Gln Pro His 35 40 45Glu Asn Val Val Ile Ser Pro His Gly Ile Ala Ser Ile Leu Gly Met 50 55 60Leu Gln Leu Gly Ala Asp Gly Arg Thr Lys Lys Gln Leu Ser Thr Val 65 70 7580 Met Arg Tyr Asn Val Asn Gly Val Gly Lys Val Leu Lys Lys Ile Asn 85 9095 Lys Ala Ile Val Ser Lys Lys Asn Lys Asp Ile Val Thr Val Ala Asn 100105 110 Ala Val Phe Val Arg Asn Gly Phe Lys Val Glu Val Pro Phe Ala Ala115 120 125 Arg Asn Lys Glu Val Phe Gln Cys Glu Val Gln Ser Val Asn PheGln 130 135 140 Asp Pro Ala Ser Ala Cys Asp Ala Ile Asn Phe Trp Val LysAsn Glu 145 150 155 160 Thr Arg Gly Met Ile Asp Asn Leu Leu Ser Pro AsnLeu Ile Asp Ser 165 170 175 Ala Leu Thr Lys Leu Val Leu Val Asn Ala ValTyr Phe Lys Gly Leu 180 185 190 Trp Lys Ser Arg Phe Gln Pro Glu Asn ThrLys Lys Arg Thr Phe Val 195 200 205 Ala Gly Asp Gly Lys Ser Tyr Gln ValPro Met Leu Ala Gln Leu Ser 210 215 220 Val Phe Arg Ser Gly Ser Thr LysThr Pro Asn Gly Leu Trp Tyr Asn 225 230 235 240 Phe Ile Glu Leu Pro TyrHis Gly Glu Ser Ile Ser Met Leu Ile Ala 245 250 255 Leu Pro Thr Glu SerSer Thr Pro Leu Ser Ala Ile Ile Pro His Ile 260 265 270 Ser Thr Lys ThrIle Asn Ser Trp Met Asn Thr Met Val Pro Lys Arg 275 280 285 Met Gln LeuVal Leu Pro Lys Phe Thr Ala Leu Ala Gln Thr Asp Leu 290 295 300 Lys GluPro Leu Lys Ala Leu Gly Ile Thr Glu Met Phe Glu Pro Ser 305 310 315 320Lys Ala Asn Phe Ala Lys Ile Thr Arg Ser Glu Ser Leu His Val Ser 325 330335 His Ile Leu Gln Lys Ala Lys Ile Glu Val Ser Glu Asp Gly Thr Lys 340345 350 Ala Ala Val Val Thr Thr Ala Ile Leu Ile Ala Arg Ser Ser Pro Pro355 360 365 Trp Phe Ile Val Asp Arg Pro Phe Leu Phe Cys Ile Arg His AsnPro 370 375 380 Thr Gly Ala Ile Leu Phe Leu Gly Gln Val Asn Lys Pro 385390 395 6 465 PRT Mus musculus 6 Met Tyr Ser Pro Gly Ala Gly Ser Gly AlaAla Gly Glu Arg Lys Leu Cys Leu Leu Ser Leu Leu Leu Ile Gly Ala Leu GlyCys Ala Ile Cys 20 25 30 His Gly Asn Pro Val Asp Asp Ile Cys Ile Ala LysPro Arg Asp Ile 35 40 45 Pro Val Asn Pro Leu Cys Ile Tyr Arg Ser Pro GlyLys Lys Ala Thr 50 55 60 Glu Glu Asp Gly Ser Glu Gln Lys Val Pro Glu AlaThr Asn Arg Arg 65 70 75 80 Val Trp Glu Leu Ser Lys Ala Asn Ser Arg PheAla Thr Asn Phe Tyr 85 90 95 Gln His Leu Ala Asp Ser Lys Asn Asp Asn AspAsn Ile Phe Leu Ser 100 105 110 Pro Leu Ser Ile Ser Thr Ala Phe Ala MetThr Lys Leu Gly Ala Cys 115 120 125 Asn Asp Thr Leu Lys Gln Leu Met GluVal Phe Lys Phe Asp Thr Ile 130 135 140 Ser Glu Lys Thr Ser Asp Gln IleHis Phe Phe Phe Ala Lys Leu Asn 145 150 155 160 Cys Arg Leu Tyr Arg LysAla Asn Lys Ser Ser Asp Leu Val Ser Ala 165 170 175 Asn Arg Leu Phe GlyAsp Lys Ser Leu Thr Phe Asn Glu Ser Tyr Gln 180 185 190 Asp Val Ser GluVal Val Tyr Gly Ala Lys Leu Gln Pro Leu Asp Phe 195 200 205 Lys Glu AsnPro Glu Gln Ser Arg Val Thr Ile Asn Asn Trp Val Ala 210 215 220 Asn LysThr Glu Gly Arg Ile Lys Asp Val Ile Pro Gln Gly Ala Ile 225 230 235 240Asn Glu Leu Thr Ala Leu Val Leu Val Asn Thr Ile Tyr Phe Lys Gly 245 250255 Leu Trp Lys Ser Lys Phe Ser Pro Glu Asn Thr Arg Lys Glu Pro Phe 260265 270 Tyr Lys Val Asp Gly Gln Ser Cys Pro Val Pro Met Met Tyr Gln Glu275 280 285 Gly Lys Phe Lys Tyr Arg Arg Val Ala Glu Gly Thr Gln Val LeuGlu 290 295 300 Leu Pro Phe Lys Gly Asp Asp Ile Thr Met Val Leu Ile LeuPro Lys 305 310 315 320 Pro Glu Lys Ser Leu Ala Lys Val Glu Gln Glu LeuThr Pro Glu Leu 325 330 335 Leu Gln Glu Trp Leu Asp Glu Leu Ser Glu ThrMet Leu Val Val His 340 345 350 Met Pro Arg Phe Arg Thr Glu Asp Gly PheSer Leu Lys Glu Gln Leu 355 360 365 Gln Asp Met Gly Leu Ile Asp Leu PheSer Pro Glu Lys Ser Gln Leu 370 375 380 Pro Gly Ile Val Ala Gly Gly ArgAsp Asp Leu Tyr Val Ser Asp Ala 385 390 395 400 Phe His Lys Ala Phe LeuGlu Val Asn Glu Glu Gly Ser Glu Ala Ala 405 410 415 Ala Ser Thr Ser ValVal Ile Thr Gly Arg Ser Leu Asn Pro Asn Arg 420 425 430 Val Thr Phe LysAla Asn Arg Pro Phe Leu Val Leu Ile Arg Glu Val 435 440 445 Ala Leu AsnThr Ile Ile Phe Met Gly Arg Val Ala Asn Pro Cys Val 450 455 460 Asn 4657 354 DNA Homo sapiens misc_feature (323) n equals a, t, g, or c 7ggaagttcct cttgctactc tggagccatt agtcaaagca cagctggttg aagaatgggc 60aaactctgtg aagaagcaaa aagtagaagt atacctgccc aggttcacag tggaacagga 120aattgattta aaagatgttt tgaaggctct tggaataact gaaattttca tcaaagatgc 180aaatttgaca ggcctctctg ataataagga gatttttctt tccaaagcaa ttcacaagtc 240cttcctagag gttaaatgaa ggaaggctcc agaagctgct gctggtcttc aggaatgatt 300tgcaattagt agggttggct gtnctgtatc cctcaaggtt attgtcggcc atcc 354 8 352DNA Homo sapiens misc_feature (60) n equals a, t, g, or c 8 agacaggaagttcctcttgc tactctggag ccattagtca aagcacagct ggttgaagan 60 tgggcaaactctgtnaagaa gcaaaaagta gaagtatacc tgcccaggtt cacagtggaa 120 caggaaattnatttaaaaga tgttttgaag gctcttggaa taactgaaat tttcatcaaa 180 gatgcaaatttgacaggcct ctctgataat aaggagattt tcntttccaa agcaattcac 240 aagtccttcctagaggttaa tgnaggaggc tccagaagct gctgctgtct cagggatgat 300 ttgcaatttangtaggntgg gctgtgctgg tatccncaag gttatttttc gg 352 9 399 DNA Homosapiens 9 ggaagttcct cttgctactc tggagccatt agtcaaagca cagctggttgaagaatgggc 60 aaactctgtg aagaagcaaa aagtagaagt atacctgccc aggttcacagtggaacagga 120 aattgattta aaagatgttt tgaaggctct tggaataact gaaattttcatcaaagatgc 180 aaatttgaca ggcctctctg ataataagga gatttttctt tccaaagcaattcacaagtc 240 cttcctagag gttaatgaag aaggctcaga agctgctgct tgtctcaggaatgattgcaa 300 ttagtaggat ggctgtgctg tatcctcaag gttattgtcg accatccatttttcctttct 360 tatcagaacc aggggacctg gtacaattct attcatggg 399 10 30 DNAArtificial Sequence primer_bind This 5′ primer sequence has a BamH1restriction site (GGATCC) followed by nucleotides encoding an aminoterminal portion of mature human BAIT BAIT (Brain-Associated Inhibitorof Tissue-Type Plasminogen Activator). 10 gagcatggat ccgccactttccctgaggaa 30 11 33 DNA Artificial Sequence primer_bind This 3′ primersequence has a BamH1 restriction site (GGATCC) and nucleotidescomplementary to the 3′ end coding sequence of the human BAIT(Brain-Associated Inhibitor of Tissue- Type Plasminogen Activator) DNAsequence. 11 gcacatggat ccttaaagtt cttcgaaatc atg 33 12 36 DNAArtificial Sequence primer_bind This 5′ primer sequence has a BamH1restriction site (GGATCC), a sequence for initiation of translation ineukaryotic cells (see Kozak, M., J. Mol. Biol. 196947-950 (1987)),followed by nucleotides encoding the amino terminus of human BAIT. 12gagcatggat ccgccatcat ggctttcctt ggactc 36 13 30 DNA Artificial Sequenceprimer_bind This 3′ primer sequence has an XbaI restriction site(TCTAGA) followed by nucleotides complementary to 3′ noncoding sequenceof the human BAIT (Brain-Associated Inhibitor of Tissue-Type PlasminogenActivator) DNA sequence. 13 gagcattcta gagttgcaaa cataatgtgc 30 14 36DNA Artificial Sequence primer_bind This 5′ primer sequence has a BamH1restriction site (GGATCC), a sequence for initiation of translation ineukaryotic cells (see Kozak, M., J. Mol. Biol. 196947-950 (1987)),followed by nucleotides encoding the amino terminus of human BAIT. 14gagcatggat ccgccatcat ggctttcctt ggactc 36 15 30 DNA Artificial Sequenceprimer_bind This 3′ primer sequence has a BamH1 restriction site(GGATCC) followed by nucleotides complementary to 3′ noncoding sequenceof the human BAIT (Brain-Associated Inhibitor of Tissue-Type PlasminogenActivator) DNA sequence. 15 gcacatggat ccaagttctt cgaaatcatg 30 16 36DNA Artificial Sequence primer_bind This 5′ primer sequence has a BamH1restriction site (GGATCC), a sequence for initiation of translation ineukaryotic cells (see Kozak, M., J. Mol. Biol. 196947-950 (1987)),followed by nucleotides encoding the amino terminus of human BAIT. 16gagcatggat ccgccatcat ggctttcctt ggactc 36 17 30 DNA Artificial Sequenceprimer_bind This 3′ primer sequence has an XbaI restriction site(TCTAGA) followed by nucleotides complementary to 3′ noncoding sequenceof the human BAIT (Brain-Associated Inhibitor of Tissue-Type PlasminogenActivator) DNA sequence. 17 gagcattcta gagttgcaaa cataatgtgc 30 18 733DNA Homo sapiens 18 gggatccgga gcccaaatct tctgacaaaa ctcacacatgcccaccgtgc ccagcacctg 60 aattcgaggg tgcaccgtca gtcttcctct tccccccaaaacccaaggac accctcatga 120 tctcccggac tcctgaggtc acatgcgtgg tggtggacgtaagccacgaa gaccctgagg 180 tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataatgccaagaca aagccgcggg 240 aggagcagta caacagcacg taccgtgtgg tcagcgtcctcaccgtcctg caccaggact 300 ggctgaatgg caaggagtac aagtgcaagg tctccaacaaagccctccca acccccatcg 360 agaaaaccat ctccaaagcc aaagggcagc cccgagaaccacaggtgtac accctgcccc 420 catcccggga tgagctgacc aagaaccagg tcagcctgacctgcctggtc aaaggcttct 480 atccaagcga catcgccgtg gagtgggaga gcaatgggcagccggagaac aactacaaga 540 ccacgcctcc cgtgctggac tccgacggct ccttcttcctctacagcaag ctcaccgtgg 600 acaagagcag gtggcagcag gggaacgtct tctcatgctccgtgatgcat gaggctctgc 660 acaaccacta cacgcagaag agcctctccc tgtctccgggtaaatgagtg cgacggccgc 720 gactctagag gat 733 19 5 PRT Homo sapiens 19Ala Thr Phe Pro Glu 1 5 20 5 PRT Homo sapiens 20 Thr Phe Pro Glu Glu 1 521 5 PRT Homo sapiens 21 Met Pro Glu Glu Ala 1 5

What is claimed is:
 1. An isolated nucleic acid molecule comprising apolynucleotide having a nucleotide sequence at least 99% identical to asequence selected from the group consisting of: (a) a nucleotidesequence encoding the BAIT polypeptide having the complete amino acidsequence in FIG. 1 (SEQ ID NO:2); (b) a nucleotide sequence encoding themature BAIT polypeptide having the amino acid sequence at positions19-410 in FIG. 1 (SEQ ID NO:2); (c) a nucleotide sequence encoding theBAIT polypeptide having the complete amino acid sequence encoded by thecDNA clone contained in ATCC Deposit No. 97722; (d) a nucleotidesequence encoding the mature BAIT polypeptide having the amino acidsequence encoded by the cDNA clone contained in ATCC Deposit No. 97722;and (e) a nucleotide sequence complementary to any of the nucleotidesequences in (a), (b), (c) or (d).
 2. The nucleic acid molecule of claimwherein said polynucleotide has the complete nucleotide sequence in FIG.1 (SEQ ID NO:1).
 3. The nucleic acid molecule of claim 1 wherein saidpolynucleotide has the nucleotide sequence in FIG. 1 (SEQ ID NO:1)encoding the BAIT polypeptide having the complete amino acid sequence inFIG. 1 (SEQ ID NO:2).
 4. The nucleic acid molecule of claim 1 whereinsaid polynucleotide has the nucleotide sequence in FIG. 1 (SEQ ID NO:1)encoding the mature BAIT polypeptide having the amino acid sequence inFIG. 1 (SEQ ID NO:2).
 5. An isolated nucleic acid molecule comprising apolynucleotide having a nucleotide sequence at least 99% identical to asequence selected from the group consisting of: (a) a nucleotidesequence encoding a polypeptide having the amino acid sequenceconsisting of residues n-410 of SEQ ID NO:2, where n is an integer inthe range of 2-49; (b) a nucleotide sequence encoding a polypeptidehaving the amino acid sequence consisting of residues 1-m of SEQ IDNO:2, where n is an integer in the range of 381-409; (c) a nucleotidesequence encoding a polypeptide having the amino acid sequenceconsisting of residues n-m of SEQ ID NO:2, where n is an integer in therange of 2-49 and m is an integer in the range of 381-409; (d) anucleotide sequence encoding a polypeptide consisting of a portion ofthe complete BAIT amino acid sequence encoded by the cDNA clonecontained in ATCC Deposit 97722 wherein said portion excludes up to 48amino acids from the amino terminus and up to 30 amino acids from theC-terminus of said complete amino acid sequence.
 6. The nucleic acidmolecule of claim 1 wherein said polynucleotide has the completenucleotide sequence of the cDNA clone contained in ATCC Deposit No.97722.
 7. The nucleic acid molecule of claim 1 wherein saidpolynucleotide has the nucleotide sequence encoding the BAIT polypeptidehaving the complete amino acid sequence encoded by the cDNA clonecontained in ATCC Deposit NO.
 97722. 8. The nucleic acid molecule ofclaim 1 wherein said polynucleotide has the nucleotide sequence encodingthe mature BAIT polypeptide having the amino acid sequence encoded bythe cDNA clone contained in ATCC Deposit No.
 97722. 9. An isolatednucleic acid molecule comprising a polynucleotide which hybridizes understringent hybridization conditions to a polynucleotide having anucleotide sequence identical to a nucleotide sequence in (a), (b), (c),(d) or (e) of claim 1 wherein said polynucleotide which hybridizes doesnot hybridize under stringent hybridization conditions to apolynucleotide having a nucleotide sequence consisting of only Aresidues or of only T residues.
 10. An isolated nucleic acid moleculecomprising a polynucleotide which encodes the amino acid sequence of anepitope-bearing portion of a BAIT polypeptide having an amino acidsequence in (a), (b ), (c) or (d) of claim
 1. 11. The isolated nucleicacid molecule of claim 10, which encodes an epitope-bearing portion of aBAIT polypeptide selected from the group consisting of: a polypeptidecomprising amino acid residues from about Val 155 to about Ala 175 (SEQID NO:2); a polypeptide comprising amino acid residues from about Phe186 to about Pro 215 (SEQ ID NO:2); a polypeptide comprising amino acidresidues from about Tyr 225 to about Ile 239 (SEQ ID NO:2); apolypeptide comprising amino acid residues from about Leu 243 to aboutLeu 255 (SEQ ID NO:2); a polypeptide comprising amino acid residues fromabout Arg 380 to about Gly 386 (SEQ ID NO:2); and a polypeptidecomprising amino acid residues from about Met 395 to about Leu 410 (SEQID NO:2).
 12. A method for making a recombinant vector comprisinginserting an isolated nucleic acid molecule of claim 1 into a vector.13. A recombinant vector produced by the method of claim
 12. 14. Amethod of making a recombinant host cell comprising introducing therecombinant vector of claim 13 into a host cell.
 15. A recombinant hostcell produced by the method of claim
 14. 16. A recombinant method forproducing a BAIT polypeptide, comprising culturing the recombinant hostcell of claim 15 under conditions such that said polypeptide isexpressed and recovering said polypeptide.
 17. An isolated BAITpolypeptide having an amino acid sequence at least 97% identical to asequence selected from the group consisting of: (a) the amino acidsequence of the BAIT polypeptide having the complete amino acid sequencein FIG. 1 (SEQ ID NO:2); (b) the amino acid sequence of the mature BAITpolypeptide having the amino acid sequence at positions 19-410 in FIG. 1(SEQ ID NO:2); (c) the amino acid sequence of the BAIT polypeptidehaving the complete amino acid sequence encoded by the cDNA clonecontained in ATCC Deposit No. 97722; (d) the amino acid sequence of themature BAIT polypeptide having the amino acid sequence encoded by thecDNA clone contained in ATCC Deposit No. 97722; and (e) the amino acidsequence of an epitope-bearing portion of any one of the polypeptides of(a), (b), (c) or (d).
 18. An isolated polypeptide comprising anepitope-bearing portion of the BAIT protein, wherein said portion isselected from the group consisting of: a polypeptide comprising aminoacid residues from about Val 155 to about Ala 175 (SEQ ID NO:2); apolypeptide comprising amino acid residues from about Phe 186 to aboutPro 215 (SEQ ID NO:2); a polypeptide comprising amino acid residues fromabout Tyr 225 to about Ile 239 (SEQ ID NO:2); a polypeptide comprisingamino acid residues from about Leu 243 to about Leu 255 (SEQ ID NO:2); apolypeptide comprising amino acid residues from about Arg 380 to aboutGly 386 (SEQ ID NO:2); and a polypeptide comprising amino acid residuesfrom about Met 395 to about Leu 410 (SEQ ID NO:2).
 19. An isolatedantibody that binds specifically to a BAIT polypeptide of claim
 17. 20.A pharmaceutical composition comprising a polypeptide of claim 17 and apharmaceutically acceptable carrier.
 21. A method of treating a patientwith the polypeptide of claim
 17. 22. The method of claim 21, whereinsaid patient has had a stroke.
 23. A method of treating a patient withthe polynucleotide of claim
 1. 24. The method of claim 23, wherein saidpatient has had a stroke.